Modulation of nuclear-retained rna

ABSTRACT

Provided herein are methods, compounds, and compositions for reducing expression of a nrRNA in an animal. Also provided herein are methods, compounds, and compositions for treating, ameliorating, delaying or reducing a symptom of a disease or disorder associated with a nuclear-retained RNA in an animal. Such methods, compounds, and compositions are useful to treat, prevent, delay, or ameliorate a disease or condition associated with a nuclear-retained RNA, or a symptom thereof.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0133WOSEQ.txt created Jul. 19, 2011, which is approximately 724 kbin size. The information in the electronic format of the sequencelisting is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Provided herein are methods of achieving a pharmacologically relevantreduction of nuclear-retained RNAs and RNAs having a long residence timein the nucleus in a tissue having low antisense oligonucleotide uptake.Such methods are useful, for example, for treating, ameliorating,delaying or reducing a symptom of a disease or disorder in an animalassociated with nuclear-retained RNAs and RNAs having a long residencetime in the nucleus.

BACKGROUND

Systemic administration of antisense oligonucleotides produces hightissue concentration in liver and renal cortex, and moderate levels insome other tissues such as adipose, spleen and certain inflammatorycells. Uptake of 2′MOE gapmer oligonucleotides in spleen andinflammatory cells is typically 2 to 5-fold less than in the liver. Inother tissues, including skeletal, smooth and cardiac muscle, tumor andbrain, systemic administration of antisense oligonucleotides results inlow to no accumulation of oligonucleotide. The systemic delivery of2′MOE gapmer oligonucleotides results in skeletal and cardiac muscleconcentration that is approximately 50-fold lower than liver. Withintissue, the oligonucleotide distribution is heterogeneous with regard tocell type. For example, glomeruli, distal tubular epithelial cells andlymphocytes demonstrate lower uptake of oligonucleotide compared toother cells in kidney and lymphoid tissue. Pharmacodynamic effects areconsistent with biodistribution data. Systemic administration of 2′MOEgapmer oligonucleotides produce modest target inhibition in skeletal orcardiac muscle in WT mice, even when targeting sequences are highlyoptimized. (Bennett C F. Pharmacological Properties of2′-O-methoxyethyl-modified oligonucleotides. In: Crooke S T, ed.Antisense Drug Technology: Principles, Strategies and Applications.2^(nd) ed. Boca Raton: CRC Press; 2008: 273-304.) Current efforts toreach targets in muscle and heart have been focused on modification ofASO chemistry or formulation and have yielded underwhelming results. Asthere are many diseases that manifest in tissues or cells that areresistant to oligonucleotide uptake, there remains a need to developmethods for effectively targeting disease associated genes in suchtissues and cells.

SUMMARY OF THE INVENTION

Provided herein are methods of achieving a pharmacologically relevantreduction of a nuclear-retained RNA. In certain embodiments, thenuclear-retained RNA is in a tissue having low antisense oligonucleotideuptake. In certain embodiments, the methods comprise administering to ananimal suspected of having said nuclear-retained RNA achemically-modified antisense oligonucleotide complementary to saidnuclear-retained RNA in an amount effective to activate a nuclearribonuclease capable of cleaving the nuclear-retained RNA in saidpharmacologically relevant amount. In certain embodiments, thenuclear-retained RNA is associated with a disease or condition in saidtissue. In certain embodiments, an animal is selected as having adisease or condition associated with a nuclear-retained RNA.

Provided herein are methods of treating, ameliorating, delaying orreducing a symptom of a disease or disorder associated with anuclear-retained RNA in a tissue having low antisense oligonucleotideuptake. In certain embodiments, the methods include selecting an animalhaving a disease or disorder associated with a nuclear-retained RNA intissue having low antisense oligonucleotide uptake. In certainembodiments, the methods include administering to an animal achemically-modified antisense oligonucleotide complementary to anuclear-retained RNA in an amount effective to activate a nuclearribonuclease capable of cleaving the nuclear-retained RNA in apharmacologically relevant amount.

In certain embodiments, the tissue is skeletal muscle, cardiac muscle,smooth muscle, adipose, spleen, bone, intestine, adrenal, testes, ovary,pancreas, pituitary, prostate, skin, uterus, bladder, tumor and brain.In certain embodiments, the cell type is cells of the glomeruli, distaltubular epithelial cells and lymphocytes.

In certain embodiments, the administering results in a systemic effectof the oligonucleotide (an effect in more than one tissue). In certainembodiments, the administering is subcutaneous, intravenous,intracerebral, intracerebroventricular, intrathecal or anotheradministration that result in a systemic effect of the oligonucleotide(an effect in more than one tissue) or delivery to the CNS or to theCSF.

Certain embodiments provide the use of any chemically-modified antisenseoligonucleotide as described herein in the manufacture of a medicamentfor use in any of the therapeutic methods described herein.

Certain embodiments provide any chemically-modified antisenseoligonucleotide as described herein, for use in any of the therapeuticmethods described herein.

Chemically-modified oligonucleotides which can be used in the methodsdescribed herein are also provided.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the animal matter described. Alldocuments, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated-by-reference forthe portions of the document discussed herein, as well as in theirentirety.

Definitions

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques can be used for chemical synthesis, andchemical analysis. Where permitted, all patents, applications, publishedapplications and other publications, GENBANK Accession Numbers andassociated sequence information obtainable through databases such asNational Center for Biotechnology Information (NCBI) and other datareferred to throughout in the disclosure herein are incorporated byreference for the portions of the document discussed herein, as well asin their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to anO-methoxy-ethyl modification of the 2′ position of a furosyl ring. A2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-O-methoxyethyl nucleotide” means a nucleotide comprising a2′-O-methoxyethyl modified sugar moiety.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position. A 5-methylcytosine is a modifiednucleobase.

“Active pharmaceutical agent” means the substance or substances in apharmaceutical composition that provide a therapeutic benefit whenadministered to an individual. For example, in certain embodiments anantisense oligonucleotide targeted to a nrRNA is an activepharmaceutical agent.

“Active target region” or “target region” means a region to which one ormore active antisense compounds is targeted. “Active antisensecompounds” means antisense compounds that reduce target nucleic acidlevels or protein levels.

“Administered concomitantly” refers to the co-administration of twoagents in any manner in which the pharmacological effects of both aremanifest in the patient at the same time. Concomitant administrationdoes not require that both agents be administered in a singlepharmaceutical composition, in the same dosage form, or by the sameroute of administration. The effects of both agents need not manifestthemselves at the same time. The effects need only be overlapping for aperiod of time and need not be coextensive.

“Administering” means providing an agent to an animal, and includes, butis not limited to, administering by a medical professional andself-administering.

“Agent” means an active substance that can provide a therapeutic benefitwhen administered to an animal. “First Agent” means a therapeuticcompound of the invention. For example, a first agent can be anantisense oligonucleotide targeting a nrRNA. “Second agent” means asecond therapeutic compound of the invention (e.g. a second antisenseoligonucleotide targeting a nrRNA) and/or a non-a nrRNA therapeuticcompound.

“Amelioration” refers to a lessening of at least one indicator, sign, orsymptom of an associated disease, disorder, or condition. The severityof indicators can be determined by animalive or objective measures,which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but notlimited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Antisense activity” means any detectable or measurable activityattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein encodedby such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable ofundergoing hybridization to a target nucleic acid through hydrogenbonding.

“Antisense inhibition” means reduction of target nucleic acid levels ortarget protein levels in the presence of an antisense compoundcomplementary to a target nucleic acid compared to target nucleic acidlevels or target protein levels in the absence of the antisensecompound.

“Antisense oligonucleotide” means a single-stranded oligonucleotidehaving a nucleobase sequence that permits hybridization to acorresponding region or segment of a target nucleic acid.

“Bicyclic sugar” means a furosyl ring modified by the bridging of twonon-geminal ring atoms. A bicyclic sugar is a modified sugar.

“Bicyclic nucleic acid” or “BNA” refers to a nucleoside or nucleotidewherein the furanose portion of the nucleoside or nucleotide includes abridge connecting two carbon atoms on the furanose ring, thereby forminga bicyclic ring system.

“Cap structure” or “terminal cap moiety” means chemical modifications,which have been incorporated at either terminus of an antisensecompound.

“Chemically distinct region” refers to a region of an antisense compoundthat is in some way chemically different than another region of the sameantisense compound. For example, a region having 2′-O-methoxyethylnucleotides is chemically distinct from a region having nucleotideswithout 2′-O-methoxyethyl modifications.

“Chimeric antisense compound” means an antisense compound that has atleast two chemically distinct regions.

“Co-administration” means administration of two or more agents to anindividual. The two or more agents can be in a single pharmaceuticalcomposition, or can be in separate pharmaceutical compositions. Each ofthe two or more agents can be administered through the same or differentroutes of administration. Co-administration encompasses parallel orsequential administration.

“Complementarity” means the capacity for pairing between nucleobases ofa first nucleic acid and a second nucleic acid.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother.

“Diluent” means an ingredient in a composition that lackspharmacological activity, but is pharmaceutically necessary ordesirable. For example, the diluent in an injected composition can be aliquid, e.g. saline solution.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration, or in a specified time period. In certainembodiments, a dose can be administered in one, two, or more boluses,tablets, or injections. For example, in certain embodiments wheresubcutaneous administration is desired, the desired dose requires avolume not easily accommodated by a single injection, therefore, two ormore injections can be used to achieve the desired dose. In certainembodiments, the pharmaceutical agent is administered by infusion overan extended period of time or continuously. Doses can be stated as theamount of pharmaceutical agent per hour, day, week, or month.

“Effective amount” or “therapeutically effective amount” means theamount of active pharmaceutical agent sufficient to effectuate a desiredphysiological outcome in an individual in need of the agent. Theeffective amount can vary among individuals depending on the health andphysical condition of the individual to be treated, the taxonomic groupof the individuals to be treated, the formulation of the composition,assessment of the individual's medical condition, and other relevantfactors.

“Fully complementary” or “100% complementary” means each nucleobase of anucleobase sequence of a first nucleic acid has a complementarynucleobase in a second nucleobase sequence of a second nucleic acid. Incertain embodiments, a first nucleic acid is an antisense compound and atarget nucleic acid is a second nucleic acid.

“Gapmer” means a chimeric antisense compound in which an internal regionhaving a plurality of nucleosides that support RNase H cleavage ispositioned between external regions having one or more nucleosides,wherein the nucleosides comprising the internal region are chemicallydistinct from the nucleoside or nucleosides comprising the externalregions. The internal region can be referred to as a “gap segment” andthe external regions can be referred to as “wing segments.”

“Gap-widened” means a chimeric antisense compound having a gap segmentof 12 or more contiguous 2′-deoxyribonucleosides positioned between andimmediately adjacent to 5′ and 3′ wing segments having from one to sixnucleosides.

“Hybridization” means the annealing of complementary nucleic acidmolecules. In certain embodiments, complementary nucleic acid moleculesinclude an antisense compound and a target nucleic acid.

“Identifying an animal with a disease or condition associated with anuclear-retained RNA” means identifying an animal having been diagnosedwith a disease or condition associated with a nuclear-retained RNA,disorder or condition or identifying an animal predisposed to develop adisease or condition associated with a nuclear-retained RNA, disorder orcondition. For example, individuals with a familial history can bepredisposed to a disease or condition associated with a nuclear-retainedRNA, disorder or condition. Such identification can be accomplished byany method including evaluating an individual's medical history andstandard clinical tests or assessments.

“Immediately adjacent” means there are no intervening elements betweenthe immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment ortherapy.

“Internucleoside linkage” refers to the chemical bond betweennucleosides.

“Linked nucleosides” means adjacent nucleosides which are bondedtogether.

“Low uptake” or “resistant to uptake” means a cell or tissue thatdemonstrate low or reduced uptake of oligonucleotide or for whichdistribution or concentration of oligonucleotide is known to be low. Incertain embodiments, a cell or tissue that has low uptake or isresistant to uptake of oligonucleotide has an oligonucleotideconcentration after systemic administration that is at least 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold,40-fold, 50-fold or 100-fold lower than liver or kidney concentration.In certain embodiments, a cell or tissue that has low uptake or isresistant to uptake of oligonucleotide demonstrates no measurablepharmacologically relevant effect upon systemic administration of anoligonucleotide targeting a non-stable (having low half-life orresidence in nucleus) or non-nuclear-retained target.

“Mismatch” or “non-complementary nucleobase” refers to the case when anucleobase of a first nucleic acid is not capable of pairing with thecorresponding nucleobase of a second or target nucleic acid.

“Modified internucleoside linkage” refers to a substitution or anychange from a naturally occurring internucleoside bond (i.e. aphosphodiester internucleoside bond).

“Modified nucleobase” refers to any nucleobase other than adenine,cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase”means the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C), and uracil (U).

“Modified nucleotide” means a nucleotide having, independently, amodified sugar moiety, modified internucleoside linkage, or modifiednucleobase. A “modified nucleoside” means a nucleoside having,independently, a modified sugar moiety or modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at leastone modified nucleotide.

“Modified sugar” refers to a substitution or change from a naturalsugar.

“Motif” means the pattern of chemically distinct regions in an antisensecompound.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage.

“Natural sugar moiety” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Non-coding RNA” or “ncRNA” means a functional RNA molecule that is nottranslated into a protein. Non-coding RNA includes highly abundant andfunctionally important RNAs such as transfer RNA (tRNA) and ribosomalRNA (rRNA), as well as RNAs such as small nucleolar RNAs (snoRNAs),microRNAs, siRNAs and piRNAs and the long ncRNAs (or large ncRNAs). LongncRNAs are generally considered to be non-protein coding transcriptslonger than about 200 nucleotides and have been shown to play roles inregulation of gene transcription, post-transcriptional regulation andepigenetic regulation (see, e.g., Guttman, M. et al., Nature., 2009,458, 223-227). Long ncRNAs may include, but are not limited to promoterdirected RNAs (pdRNAs) and lincRNAs.

“Nuclear-retained RNA” or “nrRNA” means RNA that is enriched or isstable in the nucleus. Nuclear-retained RNAs include, but are notlimited to, non-coding RNA including long ncRNA such as lincRNA, repeatelement containing RNA and expanded nucleotide repeat-containing RNA,small non-coding RNA (snRNA) including snoRNA and scaRNA (enrRNA) asdescribed herein. In certain embodiments, a nrRNA is an RNA that residesonly in the nucleus or is an RNA that pass briefly through the cytoplasmduring maturation (export to the cytoplasm followed by import back tothe nucleus) like some functional RNAs or can be an RNA that has a longresidence time in the nucleus (for example, an RNA having a longhalf-life) but is exported to the cytoplasm after a period of time orcertain event, such as, but not limited to, cleavage of a repeat element(e.g., Alu repeat element) in response to a stimulus (e.g., stress). Incertain embodiments the nuclear-retained RNA is retained within asuborganell within the nucleus. Examples of nuclear retained RNAsinclude Xlsirt, Satellite III, Hox C5 transcript variant 2 (non-coding),Menβ, Neat1, Neat2, hsr-omega, hothead, Kit, Xist, Air, Tsix, Mirg,Kcnq1ot1, AK045070, P-rex1, ZNF127AS, NESPAS, SRG1, Hotair, Gomafu,Sox2ot, Rian, CAT2, Xite, Jpx, Ftx, RoX1, RoX2, H19, Igf2, IPW, UBE3A,ATP10C, pgc, 7SK, RNA Pol II transcription elongation factor P-TEFb, B2,HSR-1, BC1, BC200, NRSE, NRON, NFAT transcription factor, Makorin-p1,HAR1F, HAR1R, OCC1, DD3/PCA3, PCGEM1, NCRMS, HIS-1, BCMS, CMPD, NC612,SRA, DISC2, PSZA11q14, RAY1/ST7, UBE3A-AS, SCA8, 22k48, C6orf37OS,COPG2IT1, DGCR5, KCNQ1 overlapping transcript 1 (non-protein coding),MESTIT 1, PRINS, SCA8/ataxin 8, ATN1/DRPLA, FMR1, AFF2/FMR2,frataxin/FXN, Htt, junctophilin-3 (JPH3), DMPK, zinc finger protein-9,Androgen receptor (AR) (X-linked), ataxin-1 (ATXN1), ATXN10, proteinphosphatase PP2A (PPP2R2B), TATA box-binding protein (TBP), ATXN2,ATXN3, CACNA1A, ATXN7, and SCA8.

“Nuclear ribonuclease” mean a ribonuclease found in the nucleus. Nuclearribonucleases include, but are not limited to, RNase H including RNaseH1 and RNase H2, the double stranded RNase drosha and other doublestranded RNases.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. Anucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids(DNA), single-stranded nucleic acids, double-stranded nucleic acids,small interfering ribonucleic acids (siRNA), and microRNAs (miRNA). Anucleic acid can also comprise a combination of these elements in asingle molecule.

“Nucleobase” means a heterocyclic moiety capable of pairing with a baseof another nucleic acid.

“Nucleobase sequence” means the order of contiguous nucleobasesindependent of any sugar, linkage, or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugaror the sugar and the base and not necessarily the linkage at one or morepositions of an oligomeric compound such as for example nucleosidemimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl,bicyclo or tricyclo sugar mimetics e.g. non furanose sugar units.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of the nucleoside.

“Nucleotide mimetic” includes those structures used to replace thenucleoside and the linkage at one or more positions of an oligomericcompound such as for example peptide nucleic acids or morpholinos(morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiesterlinkage).

“Expanded Nucleotide repeat-containing RNA” (enrRNA) means a mutant RNAmolecule that contains a sequence of nucleotides comprising an expandedrepeat element wherein a triplet or quartet of nucleotides is repeatedconsecutively several times within said sequence in greater number thannormal affecting the normal processing of said RNA (see, e.g., Cooper,T. Cell., 2009, 136, 777-793; O'Rourke, J. R., J. Biol. Chem., 2009, 284(12), 7419-7423).

“Oligomeric compound” or “oligomer” means a polymer of linked monomericsubunits which is capable of hybridizing to at least a region of anucleic acid molecule.

“Oligonucleotide” means a polymer of linked nucleosides each of whichcan be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes subcutaneousadministration, intravenous administration, intramuscularadministration, intraarterial administration, intraperitonealadministration, or intracranial administration, e.g. intrathecal orintracerebroventricular administration. Administration can becontinuous, or chronic, or short or intermittent.

“Peptide” means a molecule formed by linking at least two amino acids byamide bonds. Peptide refers to polypeptides and proteins.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual. For example, a pharmaceuticalcomposition can comprise one or more active agents and a sterile aqueoussolution.

“Pharmaceutically acceptable dose” means a dose which can provide apharmacologically relevant reduction of target and is well tolerated,having minimal, little or no toxicity.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of antisense compounds, i.e., saltsthat retain the desired biological activity of the parentoligonucleotide and do not impart undesired toxicological effectsthereto.

“Pharmacologically relevant reduction” means a reduction of an RNA thatprovides a pharmacological result. The pharmacological result can be,for example, amelioration of a disease or condition or a symptom of suchdisease or condition.

“Phosphorothioate linkage” means a linkage between nucleosides where thephosphodiester bond is modified by replacing one of the non-bridgingoxygen atoms with a sulfur atom. A phosphorothioate linkage is amodified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e. linked) nucleobasesof a nucleic acid. In certain embodiments, a portion is a defined numberof contiguous nucleobases of a target nucleic acid. In certainembodiments, a portion is a defined number of contiguous nucleobases ofan antisense compound.

“Prevent” refers to delaying or forestalling the onset or development ofa disease, disorder, or condition for a period of time from minutes toindefinitely. Prevent also means reducing risk of developing a disease,disorder, or condition.

“Prodrug” means a therapeutic agent that is prepared in an inactive formthat is converted to an active form within the body or cells thereof bythe action of endogenous enzymes or other chemicals or conditions.

“Side effects” means physiological responses attributable to a treatmentother than the desired effects. In certain embodiments, side effectsinclude injection site reactions, liver function test abnormalities,renal function abnormalities, liver toxicity, renal toxicity, centralnervous system abnormalities, myopathies, and malaise. For example,increased aminotransferase levels in serum can indicate liver toxicityor liver function abnormality. For example, increased bilirubin canindicate liver toxicity or liver function abnormality.

“Single-stranded oligonucleotide” means an oligonucleotide which is nothybridized to a complementary strand.

“Specifically hybridizable” refers to an antisense compound having asufficient degree of complementarity between an antisenseoligonucleotide and a target nucleic acid to induce a desired effect,while exhibiting minimal or no effects on non-target nucleic acids underconditions in which specific binding is desired, i.e. underphysiological conditions in the case of in vivo assays and therapeutictreatments.

“Stable RNA” means RNA with a long half-life or vary low turnover. Incertain embodiments, a stable RNA has a half-life of at least 5 hours, 6hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 20hours, 24 hours or greater than 24 hours. In certain embodiments,half-life is demonstrated by in vitro assays with RNA synthesisinhibitors such as actinomycin D or DRB (see Examples).

“Subcutaneous administration” means administration just below the skin.

“Sugar surrogate” overlaps with the slightly broader term “nucleosidemimetic” but is intended to indicate replacement of the sugar unit(furanose ring) only. The tetrahydropyranyl rings provided herein areillustrative of an example of a sugar surrogate wherein the furanosesugar group has been replaced with a tetrahydropyranyl ring system.

“Targeting” or “targeted” means the process of design and selection ofan antisense compound that will specifically hybridize to a targetnucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” allrefer to a nucleic acid capable of being targeted by antisensecompounds.

“Target segment” means the sequence of nucleotides of a target nucleicacid to which an antisense compound is targeted. “5′ target site” refersto the 5′-most nucleotide of a target segment. “3′ target site” refersto the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of an agent thatprovides a therapeutic benefit to an individual.

“Treat” refers to administering a pharmaceutical composition to effectan alteration or improvement of a disease, disorder, or condition.

“Unmodified nucleotide” means a nucleotide composed of naturallyoccurring nucleobases, sugar moieties, and internucleoside linkages. Incertain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e.β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

Certain Embodiments

Certain embodiments provide methods, compounds, and compositions forinhibiting a nuclear-retained RNA (nrRNA).

Certain embodiments provide a method of reducing a nrRNA in an animalincluding administering to the animal a compound comprising a modifiedantisense oligonucleotide targeted to the nrRNA.

In certain embodiments, the nrRNA is any of those provided herein, forexample, any one of the targets listed in Table 1 or Table 2.

In certain embodiments, the nrRNA targeted and inhibited using themethods of the invention is a nrRNA selected from Xlsirt, Satellite III,Hox C5 transcript variant 2 (non-coding), Menβ, Neat1, Neat2, hsr-omega,hothead, Kit, Xist, Air, Tsix, Mirg, Kcnq1ot1, AK045070, P-rex1,ZNF127AS, NESPAS, SRG1, Hotair, Gomafu, Sox2ot, Rian, CAT2, Xite, Jpx,Ftx, RoX1, RoX2, H19, Igf2, IPW, UBE3A, ATP10C, pgc, 7SK, RNA Pol IItranscription elongation factor P-TEFb, B2, HSR-1, BC1, BC200, NRSE,NRON, NFAT transcription factor, Makorin-p1, HAR1F, HAR1R, OCC1,DD3/PCA3, PCGEM1, NCRMS, HIS-1, BCMS, CMPD, NC612, SRA, DISC2,PSZA11q14, RAY1/ST7, UBE3A-AS, SCA8, 22k48, C6orf37OS, COPG2IT1, DGCR5,KCNQ1 overlapping transcript 1 (non-protein coding), MESTIT 1, PRINS,SCA8/ataxin 8, ATN1/DRPLA, FMR1, AFF2/FMR2, frataxin/FXN, Htt,junctophilin-3 (JPH3), DMPK, zinc finger protein-9, Androgen receptor(AR) (X-linked), ataxin-1 (ATXN1), ATXN10, protein phosphatase PP2A(PPP2R2B), TATA box-binding protein (TBP), ATXN2, ATXN3, CACNA1A, ATXN7,and SCA8. In certain embodiments, the nrRNA targeted and inhibited usingthe methods of the invention is a nrRNA selected from NEAT2 (akaMALAT1), DMPK, U16, and U 50. Certain embodiments provide a method oftreating, ameliorating, delaying or reducing a symptom of a disease ordisorder associated with a nuclear-retained RNA as described herein inan animal comprising administering to the animal a compound comprising amodified antisense oligonucleotide targeted to a nrRNA as describedherein, wherein the modified oligonucleotide reduces a nrRNA in theanimal, thereby treating, ameliorating, delaying or reducing a symptomof a disease or disorder associated with a nuclear-retained RNA in theanimal.

Certain embodiments provide a method for treating an animal with adisease or condition associated with a nuclear-retained RNA includingidentifying said animal with a disease or condition associated with anuclear-retained RNA, and administering to said animal a therapeuticallyeffective amount of a compound comprising a modified oligonucleotidetargeted to a nrRNA. In certain embodiments, the therapeuticallyeffective amount of the compound administered to the animal treats,ameliorates, delays or reduces a symptom of a disease or disorderassociated with a nuclear-retained RNA in the animal.

Certain embodiments provide a method of reducing a nrRNA comprisingadministering to an animal a compound comprising a modifiedoligonucleotide consisting of 10 to 30 linked nucleosides and having anucleobase sequence at least 90% complementary to any one of the nrRNAsequences as described herein as measured over the entirety of saidmodified oligonucleotide. In certain embodiments, the reduction in anrRNA treats, ameliorates, delays or reduces a symptom of a disease ordisorder associated with a nuclear-retained RNA in the animal.

Certain embodiments provide a method for treating, ameliorating,delaying or reducing a symptom of a disease or condition associated witha nuclear-retained RNA. The method includes identifying an animal with adisease or condition associated with a nuclear-retained RNA, andadministering to the animal a modified antisense oligonucleotidecomplementary to said nuclear-retained RNA in an amount effective toactivate a nuclear ribonuclease in a tissue resistant to or having lowuptake of oligonucleotide. The nuclear ribonuclease is capable ofcleaving the nuclear-retained RNA by recognizing the antisenseoligonucleotide/nrRNA duplex. In certain embodiments, the nuclearribonuclease is RNase H1, RNase H2 or drosha. In certain embodiments,the amount of oligonucleotide is effective to reduce thenuclear-retained RNA by a pharmacologically relevant amount. In certainembodiments, the pharmacologically relevant reduction ofnuclear-retained RNA treats, ameliorates, delays or reduces a symptom ofa disease or disorder associated with a nuclear-retained RNA in theanimal.

Certain embodiments provide a method of achieving a pharmacologicallyrelevant reduction of a nuclear-retained RNA in a tissue having lowantisense oligonucleotide uptake, comprising administering to an animalhaving or suspected of having the nuclear-retained RNA a modifiedantisense oligonucleotide targeted to or complementary to saidnuclear-retained RNA in an amount effective to activate a nuclearribonuclease capable of cleaving the nuclear-retained RNA in by apharmacologically relevant amount. In certain embodiments, thenuclear-retained RNA is associated with a disease or condition in atissue that has or demonstrates low oligonucleotide uptake or isresistant to oligonucleotide uptake and the animal is selected as havingthe associated disease or condition. In certain embodiments, thenuclear-retained RNA is a stable RNA.

Certain embodiments provide a method of treating, ameliorating, delayingor reducing a symptom of a disease or disorder associated with anuclear-retained RNA in a tissue having low antisense oligonucleotideuptake, which includes selecting an animal having a disease or disorderassociated with the nuclear-retained RNA in the tissue; andadministering to the animal a modified antisense oligonucleotidetargeted to or complementary to said nuclear-retained RNA in an amounteffective to activate a nuclear ribonuclease capable of cleaving thenuclear-retained RNA in a pharmacologically relevant amount, therebytreating, ameliorating, delaying or reducing a symptom of the disease ordisorder. In certain embodiments, the nuclear-retained RNA is a stableRNA.

In certain embodiments, the animal has a disease selected fromHuntington's diseases, Huntington's disease-like 2, myotonic dystrophy(including DM1 and DM2), fragile X-associated tremor ataxia syndrome,Fragile XE mental retardation, spinocerebellar ataxias (including thoselisted in Table 2), Friedrich's ataxia, premature ovarian insufficiency,spinal and bulbar muscular atrophy, Spinal and bulbar muscular atrophy(Kennedy's disease) or dentarubral pallidoluysian atrophy (Haw riversyndrome).

In certain embodiments, the tissue is skeletal muscle, cardiac muscle,smooth muscle, adipose, spleen, bone, intestine, adrenal, testes, ovary,pancreas, pituitary, prostate, skin, uterus, bladder, tumor and brain.In certain embodiments, the cell type is cells of the glomeruli, distaltubular epithelial cells and lymphocytes. In certain embodiments, thecell type is a malignant cell including, but not limited to, breast,lung, colon and prostate cancer cells.

In certain embodiments, the administering results in a systemic effectof the oligonucleotide (an effect in more than one tissue). In certainembodiments, the administering is subcutaneous, intravenous,intracerebral, intracerebroventricular, intrathecal or anotheradministration that result in a systemic effect of the oligonucleotide(an effect in more than one tissue) or delivery to the CNS or to theCSF.

In certain embodiments, the nrRNA is a nucleotide repeat-containing RNAcomprising at least one repeat region and at least one non-repeatregion. In certain embodiments the repeat region of said nucleotiderepeat-containing RNA comprises a repeat sequence selected from CAG,GCG, CUG, GCC, GCC, CGG, GAA, CAA, CCUG, or AUUCU. In certainembodiments, the repeat sequence is expanded. In certain embodiments,the repeat sequence repeats more than about 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 100, 250, 500, 1000, 1500, 2000 times (or any rangedefined by any two of these values) within the repeat region. In certainembodiments, the oligonucleotide targets a non-coding sequence within anon-repeat region of the nucleotide repeat-containing RNA. In certainembodiments, the oligonucleotide targets a coding region, an intron, a5′UTR, or a 3′UTR of the nucleotide repeat-containing RNA.

In certain embodiments, the nrRNA is a non-coding RNA.

In certain embodiments, the nrRNA is a long ncRNA or lincRNA.

In certain embodiments, the nrRNA is a mutant RNA. In certainembodiments, the mutant RNA is preferentially lowered compared towild-type.

In certain embodiments, the nrRNA is a stable RNA. In certainembodiments, the nrRNA has a half-life of at least 5 hours, 10 hours, 15hours, 20 hours, 24 hours, greater than 24 hours, 25 hours or greaterthan 25 hours.

In certain embodiments, the oligonucleotide is chimeric. In certainembodiments the oligonucleotide is a gapmer.

In certain embodiments, the oligonucleotide consists of 10 to 30 linkednucleosides.

In certain embodiments, the oligonucleotide has a nucleobase sequence atleast 90% complementary to the nrRNA as measured over the entirety ofsaid modified oligonucleotide. In certain embodiments, the nucleobasesequence of the modified oligonucleotide is at least 95% complementaryto any one of the nrRNAs as described herein, as measured over theentirety of said modified oligonucleotide. In certain embodiments, thenucleobase sequence of the modified oligonucleotide is 100%complementary to any one of the nrRNAs as descrived herein, as measuredover the entirety of said modified oligonucleotide.

In certain embodiments, at least one internucleoside linkage of saidmodified oligonucleotide is a modified internucleoside linkage. Incertain embodiments, each internucleoside linkage is a phosphorothioateinternucleoside linkage.

In certain embodiments, at least one nucleoside of said modifiedoligonucleotide comprises a modified sugar. In certain embodiments, atleast one modified sugar is a bicyclic sugar. In certain embodiments, atleast one modified sugar comprises a 2′-O-methoxyethyl or a4′-[C(R_(a))(R_(b))]_(n)—O-2′ bridge, wherein Ra and Rb areindependently H, alkyl or substituted alkyl. In certain embodiments, Raand Rb are each H. In certain embodiments, Ra is an alkyl and Rb is H.In certain embodiments, Ra is CH3 and Rb is H. In certain embodiments,the modified sugar comprises a 4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1or 2.

In certain embodiments, at least one nucleoside of said modifiedoligonucleotide comprises a modified nucleobase. In certain embodiments,the modified nucleobase is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide comprises: a) a gapsegment consisting of linked deoxynucleosides; b) a 5′ wing segmentconsisting of linked nucleosides; and c) a 3′ wing segment consisting oflinked nucleosides. The gap segment is positioned between the 5′ wingsegment and the 3′ wing segment and each nucleoside of each wing segmentcomprises a modified sugar.

In certain embodiments, the modified oligonucleotide comprises: a) a gapsegment consisting of ten linked deoxynucleosides; b) a 5′ wing segmentconsisting of five linked nucleosides; and c) a 3′ wing segmentconsisting of five linked nucleosides. The gap segment is positionedbetween the 5′ wing segment and the 3′ wing segment, each nucleoside ofeach wing segment comprises a 2′-O-methoxyethyl sugar, eachinternucleoside linkage of said modified oligonucleotide is aphosphorothioate linkage, and each cytosine in said modifiedoligonucleotide is a 5′-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 20linked nucleosides.

Certain embodiments provide a method of treating, ameliorating, delayingor reducing a symptom of a disease or disorder associated with anuclear-retained RNA in an animal comprising administering to the animala compound comprising a modified oligonucleotide which reduces a nrRNA,wherein the modified oligonucleotide comprises: a) a gap segmentconsisting of ten linked deoxynucleosides; b) a 5′ wing segmentconsisting of five linked nucleosides; and c) a 3′ wing segmentconsisting of five linked nucleosides. The gap segment is positionedbetween the 5′ wing segment and the 3′ wing segment, each nucleoside ofeach wing segment comprises a 2′-O-methoxyethyl sugar, eachinternucleoside linkage of said modified oligonucleotide is aphosphorothioate linkage, each cytosine in said modified oligonucleotideis a 5′-methylcytosine and said reduction of a nrRNA treats,ameliorates, delays or reduces a symptom of a disease or disorderassociated with a nuclear-retained RNA in the animal.

Certain embodiments provide the use of a compound as described herein inthe manufacture of a medicament for treating, ameliorating, orpreventing a disease or condition associated with a nuclear-retainedRNA.

In certain embodiments, the modified oligonucleotide is designated as afirst agent and the methods of the invention further compriseadministering a second agent. In certain embodiments, the first agentand the second agent are co-administered. In certain embodiments thefirst agent and the second agent are co-administered sequentially orconcomitantly.

Certain embodiments provide a kit for treating, preventing, orameliorating a disease or condition associated with a nuclear-retainedRNA as described herein wherein the kit comprises a compound asdescribed herein; and optionally an additional agent or therapy asdescribed herein. The kit can further include instructions or a labelfor using the kit to treat, prevent, or ameliorate a disease orcondition associated with a nuclear-retained RNA.

Certain embodiments provide use of a modified antisense oligonucleotideas described herein for treatment of a disease associated with anuclear-retained RNA in a tissue having low antisense oligonucleotideuptake. In certain embodiments, the use if for subcutaneous,intravenous, intracerebral, intracerebroventricular or intrathecaltreatment or treatment of the CNS or the CSF.

Certain embodiments provide the use of any chemically-modified antisenseoligonucleotide as described herein in the manufacture of a medicamentfor use in any of the therapeutic methods described herein.

Certain embodiments provide any chemically-modified antisenseoligonucleotide as described herein, for use in any of the therapeuticmethods described herein.

Certain embodiments provide a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides having anucleobase sequence comprising at least 12 contiguous nucleobases of anyof the nucleobase sequences of 92-110, 150-160, and 171-175. In certainembodiments, the modified oligonucleotide has a nucleobase sequencecomprising at least 8, at least 9, at least 10, or at least 11contiguous nucleobases of any of the nucleobase sequences of 92-110,150-160, and 171-175. In certain embodiments, the modifiedoligonucleotide has a nucleobase sequence comprising at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, or atleast 19, contiguous nucleobases of any of the nucleobase sequences of92-110, 150-160, and 171-175.

In certain embodiments, the modified oligonucleotide is asingle-stranded oligonucleotide.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is 100% complementary to SEQ ID NOs: 1, 177, and 198.

In certain embodiments, at least one internucleoside linkage is amodified internucleoside linkage.

In certain embodiments, each internucleoside linkage is aphosphorothioate internucleoside linkage.

In certain embodiments, at least one nucleoside comprises a modifiedsugar.

In certain embodiments, at least one modified sugar is a bicyclic sugar.

In certain embodiments, at least one modified sugar comprises a2′-O-methoxyethyl.

In certain embodiments, at least one nucleoside comprises a modifiednucleobase.

In certain embodiments, the modified nucleobase is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide comprises:

a gap segment consisting of linked deoxynucleosides;

a 5′ wing segment consisting of linked nucleosides;

a 3′ wing segment consisting of linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment and wherein each nucleoside of each wing segmentcomprises a modified sugar.

In certain embodiments, the modified oligonucleotide comprises:

a gap segment consisting of ten linked deoxynucleosides;

a 5′ wing segment consisting of five linked nucleosides;

a 3′ wing segment consisting of five linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar; and wherein each internucleosidelinkage is a phosphorothioate linkage.

In certain embodiments, the modified oligonucleotide consists of 14linked nucleosides.

In certain embodiments, the modified oligonucleotide consists of 16linked nucleosides.

In certain embodiments, the modified oligonucleotide consists of 20linked nucleosides.

Certain embodiments provide a pharmaceutical composition comprising acompound as described herein.

Nuclear Structures

Eukaryotic nuclei have a dynamic structural organization with severalnuclear compartments and structures. The most prominent structure is thenucleolus, a non-membrane structure in which ribosomal RNA istranscribed and processed (Thiry, M. and Lanfontaine, D. L. J. TrendsCell Biol. 2005. 15: 194-199). Chromatin domains are also importantstructures which house the genomic DNA of the cell and regulate itsexpression (Cremer, T. et al., Crit. Rev. Eukaryotic Gene Expr. 2000.10: 179-212).

The nucleus also contains speckles, or clusters of granules, of whichinterchromatin granules (ICGs) form a well-known class (Spector, D. L.et al., EMBO J. 1991. 10: 2467-3481), and which are the storage centresfor various snRNAs, snRNPs (small nuclear RNA binding proteins), and RNApolymerase II (Fu, X. Y. and Maniatis, T. Nature. 1990. 343: 437-441).Many of the larger speckles correspond to interchromatin granuleclusters (IGCs). These clusters measure 0.8-1.8 μm in diameter and arecomposed of 20-25-nm diameter particles that appear connected in places.IGCs have been proposed to be involved in the assembly and/ormodification of pre-mRNA splicing factors.

Nuclear splicing speckles or interchromatin granule clusters (IGCs) alsocontain nuclear retained RNAs (Thiry, M. Eur. J. Cell. Biol. 1993. 62:259-269). Certain nuclear retained RNAs remain in the nucleus for theduration of their life, and some of these may be released to thecytoplasm in response to certain signals, such as cellular stress. RNAis retained in the nucleus as a consequence of its editing by RNAadenosine deaminases and formation of large RNP complexes. Paraspeckles(PSs) are discrete nuclear bodies (Fox, A. H. et al., Mol. Biol. Cell2005. 16: 5304-5315) often nearby or adjacent to splicing speckles, andalso implicated in nuclear retention of certain RNAs. Studies(Clemenson, C. M. et al., Mol. Cell 2009. 33: 717-726; Sasaki, Y. T. etal., Proc. Natl. Acad. Sci. USA 2009. 106: 2525-2530; Sunwoo, H. et al.,Genome Res. 2009. 19: 347-359) have shown the two non-coding RNAs,Menε/NEAT1 and Menβ, as structural elements, critical for the formationand maintenance of PSs.

Similar to PSs are the formation of stress nuclear bodies (SNBs), whichsimilarly takes place at specific transcription sites around satelliterepeat transcripts (Denegri, M. et al., Mol. Biol. Cell 2002. 13:2069-2079; Jolly, C. et al., J. Cell Biol. 2002. 156: 775-781; Rizzi, N.et al., Mol. Cell Biol. 2004. 15: 543-551). SNBs are associated withfactors in the regulation of RNA transcription and processing, thoughtheir actual role has not yet been elucidated.

Another nuclear domain, the perichromatin fibrils (PCFs), is distributedthroughout the nucleus, and contains snRNPs, non-snRNPs and hnRNPs(Spector, D. L. Ann. Rev. Cell. Biol. 1993. 9: 265-315). These suggestthat the PCFs are the sites of transcription and pre-mRNA processing(Fakan, S. et al., J. Cell Biol. 1984. 98: 358-363). Other such activenuclear sites are coiled bodies or Cajal bodies (CBs) (Lamond, A. I. andCarmo-Fonesca, M. Trends Cell Biol. 1993. 3: 198-204; Gall, J. G. Annu.Rev. Cell Dev. Biol. 2000. 16: 273-300) that are found in cells with ahigh RNA transcriptional/processing demand (Cajal, S. R. Trab. LabInvest. Biol. Univ. Madrid 1903. 2: 129-221). CBs may represent domainsthat snRNPs would enter for maturation/recycling and leave to eventuallyintegrate other functional domains, such as the splicing speckles oractive transcriptional units.

Additional nuclear bodies are the perinuclear compartment (PNC)associated with the nucleolus and also implicated in RNA metabolism(Huang, S. J. Struct. Biol. 2000. 129: 233-240), granular and fibrillarregions of the nucleolus, promyelocytic leukemia protein (PML) nuclearbodies (Doucas, V. and Evans, R. M. Biochem. Biophys. Acta 1996. 1288:M25-M29), histone locus bodies, heat shock factor 1 foci, SAM-68 bodies,GATA-1 foci, and nuclear dots (Ascoli, C. A. and Maul, G. G. J. CellBiol. 1991. 112: 785-795).

In certain embodiments, the nrRNA targeted in the methods describedherein is contained in one of the subnuclear structures describedherein, including, but not limited to, nucleoli, Cajal bodies, speckles,paraspeckles, stress nuclear bodies, perichromatin fibrils andperinuclear compartments. In certain embodiments, the nrRNA targeted inthe methods described herein is associated with a ribonuclear protein orRNP complex.

Nuclear Retention

The cell nucleus, especially in mammalian cells, is a highly organizedstructure. Specific proteins and nucleic acids are enriched insubnuclear structures such as nucleoli, Cajal bodies, paraspeckles, andnuclear speckles (Platini and Lamond, 2004). Many of these structuresare involved in control of gene expression through retention of RNA. Themechanism of nuclear retention is thought to be mediated by a number ofstructural features of RNA. For example, inosine-containing RNAs (orRNAs that undergo adenosine to inosine hyper editing) as well as RNAswith extended 3′UTRs and RNAs with inverted repeat elements, such as Aluelements, have been linked with nuclear retention (Bond and Fox 2009).RNAs with inverted repeats elements, such as Alu repeats, are shown toform double-stranded hairpins. These hairpins associate with ribonuclearproteins and can undergo adenosine to inosine (A to I) hyper editing(Zhang and Carmichael 2001; Bond and Fox, 2009). Other studies have alsoshown the importance of A-to-I editing in human RNAs as a reason fortheir stability (Athanasiadis, 2004; Kim, 2004). Other studies haveshown that mutant expanded nucleotide repeat-containing RNAs formhairpins that associate with nuclear proteins or other proteins andthereby become sequestered or retained in the nucleus.

Thus, presence of an extended 3′ UTR, presence of expanded or invertedrepeat elements, presence of inosine or A to I editing of certainsequences and the binding of nucleoproteins to the RNA may be majorcontributing factors to nuclear retention or the increased stability ofnuclear-retained RNAs.

In certain embodiments, the nrRNA targeted or provided in the methods asdescribed herein has an extended 3′ UTR. In certain embodiments, thenrRNA has one or more inverted repeat elements. In certain embodiments,the nrRNA contains inosine or undergoes A to I editing. In certainembodiments, the nrRNA binds nucleoproteins, ribonucleoproteins orsnRNPs or a complex of any one or more thereof.

Stability of Nuclear Retained RNAs

nrRNAs are generally understood to have greater stability compared toprotein-coding RNAs. The comparative stability of nrRNAs is generallyattributed to their structure as well as association withribonucleoprotein complexes (Viegas, S. C. and Arraiano, C. M. RNA Biol.2008. 5: 230-243). For example, the MALAT1 transcript, which has a longhalf-life, has a conserved genomically encoded short poly(A)-rich tractwhich yields a short poly(A) tail-like moiety at its 3′-end in themature transcript. The presence of such short poly(A) tracts or tailshave been previously shown to be effective at ensuring RNA stability(Peng, J. et al., RNA 2005. 11: 958-965). U-rich motifs are alsobelieved to play a role in RNA stability. The presence of a poly(A)tail-like moiety and nearby U-rich motifs are believed to be the reasonfor the long half-life of MALAT-1 and its resistance to exonucleases(Wilusz J. E. et al., Cell 2008. 135: 919-932).

In certain embodiments, the nrRNA targeted or provided in the methodsdescribed herein includes a poly(A) tail or U-rich motifs.

Nuclear Retained RNAs

Non-Coding RNA

A large proportion of the mammalian genome is transcribed into longtranscripts of RNA that do not get translated into protein. Such longnon-coding RNA, previously considered as artifacts in the cell, are nowbeginning to be functionally characterized. Long non-coding RNA (longncRNA) may serve one of many functions, such as structural,house-keeping, silencing by antisense or repression, and/or regulationof other gene expression. Mercer et al. (Mercer, T. R. et al., Proc.Natl. Acad. Sci. USA 2008. 105: 716-721) have identified 849 long ncRNAsthat are expressed in the adult mouse brain and found that the majorityof processed transcripts with no protein-coding capacity functionintrinsically as RNAs. Guttman et al. (Guttman, M. et al., Nature.,2009, 458, 223-227) have identified over a thousand long ncRNAs (largeintervening non-coding RNAs or lincRNAs) and have assigned putativefunctions to each, demonstrating a diverse range of roles in processesfrom embryonic stem cell pluripotency to cell proliferation.

Long non-coding RNAs described in literature as possessing structuralfunction are Neat1 (Clemson, C. M. et al., Mol. Cell 2009. 33: 717-726),Neat2/Malat-1 (Wilusz, J. E. et al., Cell 2008. 135: 919-932), hsr-omega(Lakhotia, S. C. et al., Curr. Sci. 1999. 77: 553-563), Xlsirt, whichanchors other RNAs to the vegetal cortex in Xenopus oocytes (Kloc, M.and Etkin, L. D. Science 1994. 265: 1101-1103), Satellite III, which hasbeen implicated in the establishment and maintenance of a specificchromatin structure at the 9q12 pericentromeric region during stress(Jolly, C. and Lakhotia, S. C. Nucleic Acids Res. 2006. 34: 5508-5514),and Menβ (Sasaki, Y. T. et al., Proc. Natl. Acad. Sci. USA 2009. 106:2525-2530). Recent studies have implicated long ncRNAs as messengers ofnon-Mendelian inheritance of extragenomic information. Long ncRNAshothead in Arapidopsis thaliana (Lolle, S. J. Nature 2005. 434: 505-509)and Kit in mouse (Rassoulzadegan, M. et al., Nature 2006. 441: 469-474)both function as alternate genome caches.

Computational analysis of data from large-scale sequencing projects hasrevealed an abundance of natural antisense transcripts in eukaryoticgenomes (Lehner, B. et al., Trends Genet. 2002. 18: 63-65; Lavorgna, G.et al., Trends Biochem. Sci. 2004. 29: 88-94). More than 1600transcripts have been identified as natural antisense transcripts inhuman (Yelin, R. et al., Nat. Biotechnol. 2003. 21: 379-386). These maybe transcribed in cis or in trans. Non-coding RNAs described inliterature as possessing antisense function are Xist, which has a rolein X chromosome inactivation (Brockdorff N. et al., Cell 1992. 71:515-526, Brown, C. J. et al., Cell 1992. 71: 527-542); Tsix (Lee, J. T.et al., Nat. Gen. 1999. 21: 400-404), which is antisense to Xist; Air,which silences autosomal genes of the paternal allele (Sleutels, F. etal., Nature 2002. 415: 810-813); Cop2as, which is antisense to the Cop2gene and is maternally imprinted (Lee, Y. J. et al., FEBS Lett. 2000.472: 230-234); Mirg, antisense to the Rtl1 gene which is paternallyexpressed (Seitz, H. et al., Nat. Genet. 2003. 34: 261-262); Kcnq1ot1,an antisense transcript that regulates lineage-specific silencing(Thakur, N. et al., Mol. Cell. Biol. 2004. 24: 7855-7862; Pandey, R. R.et al., Mol. Cell 2008. 32: 232-246); AK045070, which is antisense tothe Coup-TfII gene; P-rex1AS, which is antisense to the P-rex1 gene(Mercer, T. R. et al., Proc. Natl. Acad. Sci. 2008. 105: 716-721);ZNF127AS, which is antisense to the ZNF127 gene (Jong 1999); NESPAS,antisense to the NESP55 gene (Wroe, S. F. et al., Proc. Natl. Acad. Sci.USA 2000. 97: 3342-3346); SRG1, which represses transcription of theSER3 gene in yeast (Martens, J. A. et al., Genes & Dev. 2004. 19:2695-2704); antisense transcripts against the ErbAα2 mRNA in B cells(Hastings, M. L. et al., Nucleic Acids Res. 1997. 25: 4296-4300;Hastings, M. L. et al., J. Biol. Chem. 2000. 275: 11507-11513); theantisense transcript against the HFE mRNA, which is implicated in ironmetabolism (Thenie, A. C. et al., Hum. Mol. Genet. 2001. 10: 1859-1866);the pseudo-NOS transcript, which is an antisense regulator of nNOSprotein synthesis (Korneev, S. A. J Neurosci. 1999. 19: 7711-7720); and231 known Hox ncRNAs, including Hotair, which represses transcription intrans of the HOXD locus (Rinn, J. L. et al., Cell 2007. 129: 1311-1323).

Non-coding RNAs may also regulate gene expression as dsRNAs, which alsoinduce gene silencing via RNAi pathways. Such sense-antisenseRNA-induced gene silencing has been well-documented (Aravin, A. A. Curr.Biol. 2001. 11: 1017-1027; Saito, K. et al., Genes & Dev. 2006. 20:2214-2222; Vagin, V. V. et al., Science 2006. 313: 320-324). Thesetranscripts in mammals are called piRNAs (Aravin, A. et al., Nature2006. 442: 203-207; Girard, A. et al., Nature 2006. 442: 199-202) andare involved in germline-specific transcriptional andpost-transcriptional gene silencing (Carthew, R. W. Science 2006. 313:305-306; Lau, N. C. et al., Science 2006. 313: 363-367).

Other non-coding RNAs described in literature as possessing regulatoryfunction are Evf (Feng, J. et al., Genes Dev. 2006. 20: 1470-1484),which interacts with homeobox transcription factor Dlx2; Glt2(Schuster-Gossler, K. et al., Dev. Dyn. 1998. 212: 214-228),), which isinvolved in the regulation of the dwarfism phenotype; Gomafu (Sone, M.et al., J. Cell Sci. 2007. 120: 2498-2506), which is involved in geneexpression in neurons; Sox2ot (Mikkelsen, T. S. et al., Nature 2007.448: 553-560), which contains the Sox2 gene that is an importantregulator of neurogenesis, AK021368, which encompasses mir-101a thatregulates embryo implantation, the long ncRNA bidirectional to the Satb2gene, which regulates chromatin modeling in cortical neurons, the longncRNA bidirectional to Klhl14 gene, the long ncRNA bidirectional toCamkk1 gene, which is involved in male-specific memory formation(Mercer, T. R. et al., Proc. Natl. Acad. Sci. 2008. 105: 716-721), longncRNA Rian, which is a maternally imprinted gene (Hatada, I. et al., J.Biochem. (Tokyo) 2001. 130: 187-190), CAT2 transcribed nuclear RNA,which regulates its protein-coding partner, the CAT2 gene (Prasanth, K.V. et al., Cell 2005. 123: 249-263), long ncRNAs Xite, Jpx and Ftx,which also regulates the Xist gene (Heard, E. and Disteche, C. M. Genes& Dev. 2006. 20: 1848-1867; Chureau, C. et al., Genome Res. 2002. 12:894-908), long ncRNAs RoX1 and RoX2, which regulate expression of themale X chromosome (Deng, X. and Meller, V. H. Trends Biochem. Sci. 2006.31: 526-532), long ncRNAs H19 which is a maternally imprinted gene andits corresponding paternally imprinted long ncRNA, Igf2 (Brannan, C. I.et al., Mol. Cell. Biol. 1990. 10: 28-36), the long ncRNA involved inPrader-Willi/Angelman syndrome, IPW (imprinted in Prader-Willi)(Wevrick, R. et al., Hum. Mol. Genet. 1994. 3: 1877-1882), the longncRNA UBE3A, a maternally transcribed gene (Albrecht, U. et al., Nat.Genet. 1997. 17: 75-78), and ATP10C (Meguro, M. et al., Nat. Genet.2001. 28: 19-20), long ncRNAs transcribed from the intergenic locus ofβ-globin in erythroid cells (Ashe, H. L. et al., Gens & Dev. 1997. 11:2494-2509), long ncRNAs transcribed from the intergenic locus ofIL4/IL13 loci of Th2 cells (Rogan, D. F. et al., Proc. Natl. Acad. Sci.USA. 2004. 101: 2446-2451), the miRNAs mir-iab-4-5p and mir-iab-4-3p,which regulate homeotic genes (Aravin, A. A. et al., Mol. Cell. Biol.2003. 24: 6742-6750), the long ncRNA transcripts which regulate the TRElocus (Sanchez-Elsner, T. et al., Science. 2006. 311: 1118-1123) inDrosophila, long ncRNA pgc, which is involved in germ celltranscriptional inhibition in Drosophila (Nakamura, A. et al., Science1996. 274: 2075-2079), 7SK, which binds to and inhibits RNA Pol IItranscription elongation factor P-TEFb in mammals (Nguyen, V. T. et al.,Nature 2001. 414: 322-325), long ncRNA B2 which is upregulated duringenvironmental stresses in mouse and which inhibits RNA Pol II (Allen, T.A. et al., Nat. Struct. Mol. Biol. 2004. 11: 816-821), HSR-1 whichactivates heat-shock response (Shamovsky, I. et al., Nature 2006. 440:556-560), long ncRNAs BC1 and BC200, which are targeted to the dendriticdomains in neurons and implicated in fragile X syndrome (O'Donell, W. T.and Warren, S. T. Annu. Rev. Neurosci. 2002. 25: 315-338), NRSE whichinteracts with the NRSF/REST transcriptional machinery, resulting in thetransition from neural stem cells to differentiated neurons (Kuwabara,T. et al., Cell 2004. 116: 779-793), viral long ncRNAs EBER1 and EBER2,which are expressed during viral latency in viruses like Epstein-Barrvirus (Lerner, M. R. et al., Proc. Natl. Acad. Sci. USA 1981. 78:805-809), and which also play a key role in the maintenance of themalignant phenotype of Burkitt's lymphoma cells (Nanbo, A. and Takada,K. Rev. Med. Virol. 2002. 12: 321-326), NRON, which is a repressor ofthe NFAT transcription factor (Willingham, A. T. et al., Science 2005.309: 1570-1573), Makorin-p1, which stabilizes Makorin-1 mRNA (Yano, Y.et al., J. Mol. Med. 2004. 82: 414-422), long ncRNAs HAR1F and HAR1R,both of which are implicated in human brain evolution (Pollard, K. S. etal., Nature 2006. 443: 167-172), and the long ncRNAs PROMPTs, which areproduced upstream of active transcription sites and which positivelycorrelated to gene activity (Preker, P. et al., Science 2008. 322:1851-1854).

Expression analyses comparing tumor cells with normal cells havedemonstrated changes in the expression levels of certain long ncRNAs inseveral forms of cancer. The long ncRNAs reported are H19 in pediatriccancers (DeBaun, M. R. et al., Am. J. Hum. Genet. 2002. 70: 604-611),and Igf2 in Wilms' tumor and several fetal tumors (Okutsu, T. et al., J.Biochem. 2000. 127: 475-483), both of which have reduced expression inthe tumor cells, OCC1, which is overexpressed in colon carcinoma(Pibouin, L. et al., Cancer Genet. Cytogenet. 2002. 133: 55-60), longncRNAs DD3/PCA3 and PCGEM1, which are overexpressed in prostate tumors(Bussemakers, M. J. et al., Cancer Res. 1999. 59: 5975-5979), MALAT-1,which is overexpressed in non-small-cell lung cancer (Ji, P. et al.,Oncogene 2003. 22: 8031-8041) and in uterine endometrial stromal sarcomaand hepatocellular carcinoma (Yamada, K. et al., Cancer Sci. 2006. 97:106-112), NCRMS, which is overexpressed in alveolar rhabdomyosarcoma(Chan, A. S. et al., Oncogene 2002. 21: 3029-3037), HIS-1, which isimplicated in lymphomagenesis and erythroleukemogenesis (Tam, W. et al.,J. Virol. 2002. 76: 4275-4286), BC200, which is overexpressed in breast,cervix, esophagus, lung, ovary, parotid, and tongue cancers (Chen, W. etal., J. Pathol. 1997. 183: 345-351), BCMS, which is implicated in B-cellneoplasia (Wolf, S. et al., Hum. Mol. Genet. 2001. 10: 1275-1285), CMPD,which is implicated in Campomyelic dysplasia (Ninomiya, S. et al., Hum.Mol. Genet. 1996. 5: 69-72), HOST2, which is expressed in ovarian cancercells (Rangel, L. B. et al., Oncogene 2003. 22: 7225-7232), NC612, whichis implicated in prostate cancer (Silva, A. P. et al., Gene 2003. 310:49-57), SRA, which is a steroid receptor activated RNA isoform expressedin breast cancer (Lanz, R. B. et al., Cell 1999. 97: 17-27), and TRNG10,which is implicated in various cancers (Roberts, T. et al., Hum. Mol.Genet. 1998. 7: 1169-1178).

Other long ncRNAs implicated in various disorders are BC200 inAlzheimer's disease (Lukiw, W. J. et al., Neurochem. Res. 1992. 17:591-597), DISC2 in schizophrenia and bipolar affective disorder (Millar,J. K. et al., Hum. Mol. Genet. 2000. 9: 1415-1423; Millar J. K. et al.,Ann. Med. 2004. 36: 367-378), IPW in Prader-Willi syndrome (Wevrick, R.et al., Hum. Mol. Genet. 1994. 3: 1877-1882), prion-associated RNAs inprion pathologies (Deleault, N. R. et al., Nature. 2003. 425: 717-720),PSZA11q14, which has reduced expression in the brains of schizophrenicpatients (Polesskaya, O. O. et al., J. Neurosci. Res. 2003. 74:111-122), RAY1/ST7 in autistic disorder (Vincent, J. B. et al., Genomics2002. 80: 283-294), SCA8 in spinocerebellar ataxia type 8 (Nemes, J. P.et al., Hum. Mol. Genet. 2000. 9: 1543-1551), UBE3A-AS in Angelmansyndrome (Chamberlain, S. J. and Brannan, C. I. Genomics 2001. 73:316-322), ZNF127AS in Prader-Willi syndrome (Jong, M. T. et al., Hum.Mol. Genet. 1999. 8: 783-793), 22k48 in DiGeorge syndrome (Pizzuti, A.et al., Mol. Genet. Metab. 1999. 67: 227-235), C6orf37OS in diffusepanbronchiolotis (Matsuzaka, Y. et al., Immunogenetics 2002. 54:301-309), COPG2IT1 in Russell-Silver syndrome (Yamasaki, K. et al.,Genomics 2000. 68: 330-335), DGCR5, which is disrupted in DiGeorgesyndrome (Sutherland, H. F. et al., Am. J. Hum. Genet. 1996. 59: 23-31),H19 and LIT1 in Beckwith-Wiedemann syndrome (Sparago, A. et al., Nat.Genet. 2004. 36: 958-960; Niemitz, E. L. et al., Am. J. Hum. Genet.2004. 75: 844-849), LIT1 in Romano-Ward, Jervell and Lange-Nielsensyndromes (Horike, S. et al., Hum. Mol. Genet. 2000. 9: 2075-2083),MESTIT 1 in Russell-Silver syndrome (Li, T. et al., J. Biol. Chem. 2002.277: 13518-13527), and PRINS in psoriasis (Sonkoly, E. et al., J. Biol.Chem. 2005. 280: 24159-24167).

Certain non-coding pri-miRNA precursors in the nucleus are alsoassociated with disease and can be targeted by the methods providedherein. For example, BIC is a nuclear non-coding pri-miRNA precursorwhich is overexpressed in non-Hodgkin's lymphoma and Burkitt's lymphomas(van den Berg, A. et al., Genes Chromosomes Cancer 2003. 37: 20-28).

Small nucleolar RNAs (snoRNA) are another type of nuclear-retainednon-coding RNA localized to the nucleolus inside the nucleus ofeukaryotic cells. In certain instances, such snoRNA have been shown tobe associated with precursors of ribosomal RNA (rRNA). Accordingly,certain snoRNAs have been reported to be involved in nucleotidemodification and processing of pre-rRNA. The snoRNA U16 and U50 functionin the modification of other small nuclear RNAs (Fragapane, P. et al.,EMBO J. 12: 2921-2928, 1993; Tanaka, R. et al., Genes Cells. 5: 277-287,2000). Nucleic acids have also been found in Cajal bodies within thenucleus. RNA found in Cajal bodies have been referred to as small Cajalbody-specific RNA (scaRNA). Certain scaRNA have been reported to beinvolved in nucleotide modification of spliceosomal small nuclear RNAs(snRNAs). SnoRNA machinery has been implicated in human diseases such asDyskeratosis congenital and Prader-Willi syndrome (Meier, U. T.,Chromosoma 2005 114: 1-14).

TABLE 1 Gene ID/ Gene Accession Other Aliases SEQ ID NO Xlsirt S67412 1Satellite III X06137.1 SatIII, SatIII - repeat containing 2 RNAs Hox C5transcript NR_003084 Home box C5, mir-615 3 variant 2 (non- coding) MenβGQ859162.1 4 Neat1 NR_002802.2 Men ε 5 Neat2 NR_002819.2 Malat-1,metastasis associated 6 lung adenocarcinoma transcript 1 (non-proteincoding) hsr-omega NR_002068.3 Hsromega; Heat shock RNA 7 omega hotheadNM_001160997.1 HTH HTH (HOTHEAD); FAD 8 binding/aldehyde-lyase/mandelonitrile lyase Kit NM_000222.1 KIT; v-kit Hardy-Zuckerman 4 9feline sarcoma viral oncogene homolog Xist NR_001564 X(inactive)-specific transcript 10 (non-protein coding) Air DQ220014.1air; aerotaxis receptor protein 11 Tsix NR_002844.2 X(inactive)-specific transcript, 12 antisense Mirg NR_028265.1 Mirg miRNAcontaining gene 13 Kcnq1ot1 NR_002728 KCNQ1 overlapping transcript 1 14AK045070 AK045070 B130024G19Rik RIKEN cDNA 15 RIKEN B130024G19 geneP-rex1 NM_020820 phosphatidylinositol-3,4,5- 16 trisphosphate-dependentRac exchange factor 1 ZNF127AS AF130844.1 MKRN3AS; MKRN3 antisense 17RNA (non-protein coding) NESPAS AJ251759 neuroendocrine secretoryprotein 18 antisense SRG1 NM_202885.2 SRG1; (SENESCENCE- 19 RELATED GENE1 Hotair NR_003716 hox transcript antisense RNA 20 (non-protein coding)Gomafu NR_033657 Miat; myocardial infarction 21 associated transcript(non-protein coding) Sox2ot NR_004053 SOX2OT; SOX2 overlapping 22transcript (non-protein coding) Rian NR_028261.1 Rian; RNA imprinted and23 accumulated in nucleus CAT2 U35654.1 mCat2; cationic amino acid 24transporter Xite AY190762.1 Xite; X-inactivation intergenic 25transcription element Jpx AV714079 NCRNA00183 non-protein 26 coding RNA183 Ftx NR_028379 FTX; Thrombocytosis, familial 27 X-linked, FLJ18387,NCRNA00182 RoX1 NR_002098.1 roX1; RNA on the X 1 28 RoX2 NR_002105.1roX2; RNA on the X 2 29 H19 NR_002196.1 H19; H19, imprinted maternally30 expressed transcript (non-protein coding) Igf2 NM_000612 Igf2;insulin-like growth factor 2 31 IPW U12897 IPW; imprinted inPrader-Willi 32 syndrome (non-protein coding) UBE3A NM_000462 Ube3a;ubiquitin protein ligase 33 E3A ATP10C NM_024490 ATP10C; ATPase, ClassV, type 34 10C pgc NM_001103942.1 pgc; polar granule component 35 7SKNR_001445.2 RN7SK RNA, 7SK small nuclear 36 RNA Pol II AF027300.1Positive transcription elongation 37 transcription factor b elongationfactor P-TEFb B2 B2 hypothetical protein HSR-1 U08215.1 HSR1 similar toCandida 39 troplicalis heat-shock related protein Hsr1p that conferssalt tolerance to S. cerevisiae; potential HSF-type DNA bindingtranscription factor BC1 NR_033762.2 Brain cytoplasmic RNA 1 40 BC200NR_001568.1 BCYRN1 brain cytoplasmic RNA 41 1 (non-protein coding) NRSEAX934359.1 neuron-restrictive silencer 42 element NRON AK042215.1non-protein coding RNA, 43 repressor of NFAT NFAT NM_012340.3 Rel NFATtranscription factor 44 transcription factor Makorin-p1 NM_013446 Mkrn1makorin, ring finger 45 protein, 1 HAR1F NR_003244.1 HAR1A highlyaccelerated region 46 1A (non-protein coding) HAR1R NR_003245 HAR1Bhighly accelerated region 47 1B (non-protein coding) OCC1 AB039661.11500009L16Rik RIKEN cDNA 48 1500009L16 gene DD3/PCA3 NR_015342 PCA3prostate cancer antigen 3 49 (non-protein coding) PCGEM1 NR_002769PCGEM1 prostate-specific 50 transcript 1 (non-protein coding) NCRMSNR_024037 RMST rhabdomyosarcoma 2 51 associated transcript (non-proteincoding) HIS-1 U09772.1 His1 hematopoietic insertion site 1 52 BCMSNR_002605 BCMS hypothetical LOC647154, 53 DLB1 CMPD cmpD bicarbonatetransport system ATP-binding protein NC612 AF510427 Homo sapiens cloneNC612 55 noncoding mRNA sequence SRA NM_001035235.2 sra; sarah 56 DISC2NR_002227 DISC2 disrupted in schizophrenia 57 2 (non-protein coding)PSZA11q14 AF525782 DLG2AS DLG2 antisense RNA 58 (non-protein coding)RAY1/ST7 NM_021908 ST7 suppression of 59 tumorigenicity 7 UBE3A-ASNG_002690 Ube3a ubiquitin protein ligase E3A SCA8 NR_002717 sca8 cellsurface antigen-like 61 protein Sca8, KLHL1AS, ATXN8OS ZNF127ASAF130844.1 MKRN3AS MKRN3 antisense 62 RNA (non-protein coding) 22k48AF093016 Homo sapiens 22k48 gene, 63 5′UTR C6orf37OS NM_080870 DPCR1diffuse panbronchiolitis 64 critical region 1, PBLT, bCX105N19.6COPG2IT1 NR_024086 COPG2IT1 COPG2 imprinted 65 transcript 1 (non-proteincoding) DGCR5 NR_002733 DGCR5 DiGeorge syndrome 66 critical region gene5 (non-protein coding) KCNQ1 NR_002728 KvDMR1, KvLQT1-AS, LIT1, 67overlapping NCRNA00012, transcript 1 (non- protein coding) MESTIT 1AF482998 Homo sapiens MESTIT1 68 antisense RNA, partial sequence PRINSNR_023388 PRINS psoriasis associated RNA 69 induced by stress(non-protein coding), NCRNA00074

Expanded Nucleotide Repeat-Containing RNAs

Mutant expanded nucleotide repeat-containing RNAs can form hairpins thatassociate with nuclear proteins or other proteins that becomesequestered or retained in the nucleus. These expanded nucleotiderepeat-containing RNAs (enrRNAs) are also referred to in the art as“gain-of-function RNAs” that gain the ability to sequester ribonuclearproteins and impair the normal action of RNA processing in the nucleus(see Cooper, T. (2009) Cell 136, 777-793; O'Rourke, J R (2009) J. Biol.Chem. 284 (12), 7419-7423). Several disease states are associated withenrRNAs, some of said diseases only occurring where a threshold numberof repeats are contained within the enrRNA. For instance, one diseasestate might be caused by 50-200 repeats in a particular gene, where adifferent disease or severity is caused by a different number ofrepeats >400 in the same gene. Some mutations that caused enrRNAs can beheterozygous and therefore some copies of the gene can be functional andas a result, there is a need to interfere with the mutant version of thegene without affecting the wild type copy of the gene. Examples ofnucleotide repeat-containing RNA molecules that can have expanded repeatelements implicated in disease are the following:

TABLE 2 COPY COPY SEQ AFFECTED NUMBER NUMBER GENBANK ID DISEASE REPEATGENE (NORMAL) (DISEASED) Reference Accession No NO Ataxin 8 CUG with orSCA8/ataxin 8 16-37 107-127 Nat. Genet 21: NG_016173.1 70 oppositewithout 379, 1999 strand interruptions (NCBI/OMIM) (ATXN8OS) Atrophin 1CAG ATN1/DRPLA  7 to 34  49-93 Nat. Genet. 10: NM_001007026.1 71 (DRPLA)99, 1995 Fragile X- CGG FMR1 200-4500 Annu. Rev. NM_002024.5 72associated Neurosci. 25: tremor/ataxi 315, 2002 a syndrome <55 >200 Am.J Hum. (FXTAS) Genet. 72: 869, 2003 (NCBI/OMIM) Fragile XE GCC AFF2/FMR2 6 to 25 >200 Am. J. Hum. AB102644.1 73 mental Genet. 55: 81,retardation 1994 (FRAXE) (NCBI/OMIM) Friedreich's GAA frataxin/FXN  5 to30  70 to >1000 Genomics 88: BC048097.1 74 ataxia 580, 2006 (FRDA)(NCBI/OMIM) Huntington CAG Htt <28 >36 Lancet 369: 220, NM_002111.6 75disease 2007 Huntington CAG/CUG junctophilin-3  6 to 28  44 to 57 Nat.Clin Prac AB042636.1 76 disease-like (JPH3) Neurol. 3: 517, 2 (HDL2)2007 Myotonic CUG DMPK  5 TO 35  80 TO >2500 Harper, L19268.1 77dystrophy Myotonic (DM1) Dystrophy (Saunders, London, ed.3, 2001)  50to >3500 Annu. Rev. Neurosci. 29: 259, 2006  5 to 37 >50 EMBO J. 19:4439, 2000  50 to >2000 Curr Opin Neurol. 20: 572, 2007 DM2 CCUG zincfinger  75 to 11,000 Science 293: NM_011763.2 78 protein-9 864, 2001(NCBI/OMIM) Spinal and CAG Androgen 10 to 36  38 to 62 Nature 352: 77,M20132.1 79 bulbar receptor (AR) 1991 muscular (X-linked) atrophy/Kennedy disease Spinocerebellar CAG ataxin-1  6 to 35  49 to 88NCBI/OMIM NM_000332.3 80 ataxia 1 (ATXN1) Spinocerebellar AUUCU ATXN1010 to 29 280 to 4500 Neurology 66: BC007508.2 81 ataxia 10 1602, 2006(NCBI/OMIM) Spinocerebellar CAG protein  9 to 28  55 to 78 Brain ResBull. NM_004576.2 82 ataxia 12 phosphatase 56: 397, 2001 PP2A (PPP2R2B) 7 to 28  66 to 78 Wikipedia Spinocerebellar CAG TATA box- 25 to 42  47to 63 Eur. J. Hum. M55654.1 83 ataxia 17/ binding Genet. 9: 160,Huntington protein (TBP) 2001 disease- (NCBI/OMIM) like 4 (HDL4)Spinocerebellar CAG ATXN2 17 to 29  37 to 50 Nat. Genet. 14: NM_002973.384 ataxia 2 285, 1996 (NCBI/OMIM) 15 to 34  35 to 59 Nat. Genet. 14:277, 1996 (NCBI/OMIM) 14 to 32  33 to 77 Wikipedia Spinocerebellar CAGATXN3 10 to 51  55-87 Human Mol. AB050194.1 85 ataxia 3 Genet. 17: 2071,(Machado- 2008 Joseph (NCBI/OMIM) disease 12 to 40  55 to 86 WikipediaSpinocerebellar CAG CACNA1A  4 to 18  21 to 30 Wikipedia FJ040507.1 86ataxia 6  5 to 20  21 to 25 Am. J. Hum. Genet. 61: 336, 1997 (NCBI/OMIM)Spinocerebellar CAG ATXN7  7 to 17  38-130 Nat. Genet. 17: AJ000517.1 87ataxia 7/ 65, 1997 OPCA3 (NCBI/OMIM) Spinocerebellar CUG SCA8  74to >1300 Nat. Genet. 21: AF126749.1 88 ataxia 8 379, 1999

An example from the table is myotonic dystrophy type 1 (DM1). DM1, withan estimated frequency of 1 in 7,500, is an autosomal dominant diseasethat causes progressive disability and premature death. Skeletal,cardiac, and smooth muscle are affected. No treatment has been shown tomodify the course of this disorder. The cause of DM1 is an expansion ofCTG repeats in the 3′ untranslated region (UTR) of DMPK, a gene encodinga cytosolic protein kinase. The mutation is unstable in dividing andpost-mitotic cells, with a bias towards further expansion. Affectedindividuals typically have DMPK alleles with several thousand repeats inskeletal and cardiac muscle.

Research on DM1 has led to the discovery of RNA dominance, a diseaseprocess in which expression of RNA containing an expanded CUG repeat(CUGexp) induces cell dysfunction and ultimately cell degeneration inmuscle. A critical step in this process is the interaction of CUGrepeats with splicing factors in the Muscleblind-like (MBNL1) family.This interaction causes retention of CUGexp RNA in nuclear foci, whichadversely effects transcriptional and post-transcriptional regulation ofother genes. Treatment of the disease is complicated because the drug oragent must be taken up by muscle tissue. Oligonucleotide uptake inmuscle tissue is very low.

Reduction of Nuclear-Retained RNA

Data provided herein demonstrates that sensitivity to cleavage by ASOsis dramatically increased for a nuclear-retained RNA making it possibleto reduce nuclear-retained targets in tissue that has low uptake ofoligonucleotide by a pharmacologically relevant amount. For example, outof the more than 4,000 transcripts that Isis has targeted by antisense,MALAT1, a non-coding, nuclear-retained RNA, is demonstrated to be one ofthe most sensitive targets for antisense oligonucleotide/RNase Hinhibition. The data demonstrate a great number of oligonucleotidestargeting over the majority of the transcript that inhibit by more than50% in vitro. The data also demonstrates very low IC50 values inmultiple cell types. Half-life studies have also shown that the MALAT1is stable over a period of at least 10 hours. Subcutaneousadministration of oligonucleotide targeting MALAT1 at doses commensuratewith other oligonucleotide drugs (e.g., liver targeting drugs) achievedpharmacologically relevant reduction of MALAT1 in skeletal and cardiacmuscle. Dosing at 50 mg/kg biweekly for 3.5 weeks achieved a 89% and 85%reduction in gastrocnemius and quadriceps, respectively, and 54%reduction in heart (as compared to 95% reduction in liver).Pharmacologically relevant reduction of MALAT1 has also been achieved intumor xenograft models.

Where the nuclear-retained RNA is a mutant RNA, this sensitivity alsoprovides a means to selectively reduce mutant RNA over wild-type. Thisapproach is advantageous as the risk of inducing functional proteindeficiency is reduced.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics,antisense compounds, antisense oligonucleotides, and siRNAs. Anoligomeric compound can be “antisense” to a target nucleic acid, meaningthat is capable of undergoing hybridization to a target nucleic acidthrough hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequencethat, when written in the 5′ to 3′ direction, comprises the reversecomplement of the target segment of a target nucleic acid to which it istargeted. In certain such embodiments, an antisense oligonucleotide hasa nucleobase sequence that, when written in the 5′ to 3′ direction,comprises the reverse complement of the target segment of a targetnucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to a nrRNAnucleic acid is 10 to 30 nucleotides in length. In other words,antisense compounds are from 10 to 30 linked nucleobases. In otherembodiments, the antisense compound comprises a modified oligonucleotideconsisting of 8 to 80, 10-80, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or20 linked nucleobases. In certain such embodiments, the antisensecompound comprises a modified oligonucleotide consisting of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linkednucleobases in length, or a range defined by any two of the abovevalues.

In certain embodiments, the antisense compound comprises a shortened ortruncated modified oligonucleotide. The shortened or truncated modifiedoligonucleotide can have a single nucleoside deleted from the 5′ end (5′truncation), or alternatively from the 3′ end (3′ truncation). Ashortened or truncated oligonucleotide can have two nucleosides deletedfrom the 5′ end, or alternatively can have two subunits deleted from the3′ end. Alternatively, the deleted nucleosides can be dispersedthroughout the modified oligonucleotide, for example, in an antisensecompound having one nucleoside deleted from the 5′ end and onenucleoside deleted from the 3′ end.

When a single additional nucleoside is present in a lengthenedoligonucleotide, the additional nucleoside can be located at the 5′ or3′ end of the oligonucleotide. When two or more additional nucleosidesare present, the added nucleosides can be adjacent to each other, forexample, in an oligonucleotide having two nucleosides added to the 5′end (5′ addition), or alternatively to the 3′ end (3′ addition), of theoligonucleotide. Alternatively, the added nucleoside can be dispersedthroughout the antisense compound, for example, in an oligonucleotidehaving one nucleoside added to the 5′ end and one subunit added to the3′ end.

It is possible to increase or decrease the length of an antisensecompound, such as an antisense oligonucleotide, and/or introducemismatch bases without eliminating activity. For example, in Woolf etal. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series ofantisense oligonucleotides 13-25 nucleobases in length were tested fortheir ability to induce cleavage of a target RNA in an oocyte injectionmodel. Antisense oligonucleotides 25 nucleobases in length with 8 or 11mismatch bases near the ends of the antisense oligonucleotides were ableto direct specific cleavage of the target mRNA, albeit to a lesserextent than the antisense oligonucleotides that contained no mismatches.Similarly, target specific cleavage was achieved using 13 nucleobaseantisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001)demonstrated the ability of an oligonucleotide having 100%complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xLmRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and invivo. Furthermore, this oligonucleotide demonstrated potent anti-tumoractivity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a seriesof tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42nucleobase antisense oligonucleotides comprised of the sequence of twoor three of the tandem antisense oligonucleotides, respectively, fortheir ability to arrest translation of human DHFR in a rabbitreticulocyte assay. Each of the three 14 nucleobase antisenseoligonucleotides alone was able to inhibit translation, albeit at a moremodest level than the 28 or 42 nucleobase antisense oligonucleotides.

Antisense Compound Motifs

In certain embodiments, antisense compounds targeted to a nrRNA nucleicacid have chemically modified subunits arranged in patterns, or motifs,to confer to the antisense compounds properties such as enhanced theinhibitory activity, increased binding affinity for a target nucleicacid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. A second region of achimeric antisense compound can optionally serve as a substrate for thenuclear ribonuclease RNase H, which cleaves the RNA strand of an RNA:DNAduplex.

Antisense compounds having a gapmer motif are considered chimericantisense compounds. In a gapmer an internal region having a pluralityof nucleotides that supports RNaseH cleavage is positioned betweenexternal regions having a plurality of nucleotides that are chemicallydistinct from the nucleosides of the internal region. In the case of anantisense oligonucleotide having a gapmer motif, the gap segmentgenerally serves as the substrate for endonuclease cleavage, while thewing segments comprise modified nucleosides. In certain embodiments, theregions of a gapmer are differentiated by the types of sugar moietiescomprising each distinct region. The types of sugar moieties that areused to differentiate the regions of a gapmer can in some embodimentsinclude β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modifiednucleosides (such 2′-modified nucleosides can include 2′-MOE, and2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (suchbicyclic sugar modified nucleosides can include those having a4′-(CH2)n-O-2′ bridge, where n=1 or n=2). Preferably, each distinctregion comprises uniform sugar moieties. The wing-gap-wing motif isfrequently described as “X-Y-Z”, where “X” represents the length of the5′ wing region, “Y” represents the length of the gap region, and “Z”represents the length of the 3′ wing region. As used herein, a gapmerdescribed as “X-Y-Z” has a configuration such that the gap segment ispositioned immediately adjacent each of the 5′ wing segment and the 3′wing segment. Thus, no intervening nucleotides exist between the 5′ wingsegment and gap segment, or the gap segment and the 3′ wing segment. Anyof the antisense compounds described herein can have a gapmer motif. Insome embodiments, X and Z are the same, in other embodiments they aredifferent. In a preferred embodiment, Y is between 8 and 15 nucleotides.X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides. Thus, gapmersinclude, but are not limited to, for example 5-10-5, 4-8-4, 4-12-3,4-12-4, 3-14-3, 2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2,6-8-6, 5-8-5, 1-8-1, or 2-6-2.

In certain embodiments, the antisense compound as a “wingmer” motif,having a wing-gap or gap-wing configuration, i.e. an X-Y or Y-Zconfiguration as described above for the gapmer configuration. Thus,wingmer configurations include, but are not limited to, for example5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, or5-13.

In certain embodiments, antisense compounds targeted to a nrRNA nucleicacid possess a 5-10-5 gapmer motif.

In certain embodiments, an antisense compound targeted to a nrRNAnucleic acid has a gap-widened motif.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode a nrRNA include, without limitation,those described herein, including those listed in Table 1 and Table 2.

It is understood that the sequence set forth in each SEQ ID NO in theExamples contained herein is independent of any modification to a sugarmoiety, an internucleoside linkage, or a nucleobase. As such, antisensecompounds defined by a SEQ ID NO can comprise, independently, one ormore modifications to a sugar moiety, an internucleoside linkage, or anucleobase. Antisense compounds described by Isis Number (Isis No)indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined regionof the target nucleic acid. For example, a target region can encompass a3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region. The structurally defined regionsfor a nrRNA can be obtained by accession number from sequence databasessuch as NCBI and such information is incorporated herein by reference.In certain embodiments, a target region can encompass the sequence froma 5′ target site of one target segment within the target region to a 3′target site of another target segment within the target region.

Targeting includes determination of at least one target segment to whichan antisense compound hybridizes, such that a desired effect occurs. Incertain embodiments, the desired effect is a reduction in mRNA targetnucleic acid levels. In certain embodiments, the desired effect isreduction of levels of protein encoded by the target nucleic acid or aphenotypic change associated with the target nucleic acid.

A target region can contain one or more target segments. Multiple targetsegments within a target region can be overlapping. Alternatively, theycan be non-overlapping. In certain embodiments, target segments within atarget region are separated by no more than about 300 nucleotides. Incertain embodiments, target segments within a target region areseparated by a number of nucleotides that is, is about, is no more than,is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30,20, or 10 nucleotides on the target nucleic acid, or is a range definedby any two of the preceding values. In certain embodiments, targetsegments within a target region are separated by no more than, or nomore than about, 5 nucleotides on the target nucleic acid. In certainembodiments, target segments are contiguous. Contemplated are targetregions defined by a range having a starting nucleic acid that is any ofthe 5′ target sites or 3′ target sites listed herein.

Suitable target segments can be found within a 5′ UTR, a coding region,a 3′ UTR, an intron, an exon, or an exon/intron junction. Targetsegments containing a start codon or a stop codon are also suitabletarget segments. A suitable target segment can specifically exclude acertain structurally defined region such as the start codon or stopcodon.

The determination of suitable target segments can include a comparisonof the sequence of a target nucleic acid to other sequences throughoutthe genome. For example, the BLAST algorithm can be used to identifyregions of similarity amongst different nucleic acids. This comparisoncan prevent the selection of antisense compound sequences that canhybridize in a non-specific manner to sequences other than a selectedtarget nucleic acid (i.e., non-target or off-target sequences).

There can be variation in activity (e.g., as defined by percentreduction of target nucleic acid levels) of the antisense compoundswithin an active target region. In certain embodiments, phenotypicchanges, such as a treating, ameliorating, delaying or reducing asymptom of a disease or disorder associated with a nuclear-retained RNA,are indicative of inhibition of a nrRNA.

Hybridization

In some embodiments, hybridization occurs between an antisense compounddisclosed herein and a nrRNA. The most common mechanism of hybridizationinvolves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen bonding) between complementary nucleobases of thenucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditionsare sequence-dependent and are determined by the nature and compositionof the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art (Sambrooke andRussell, Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). Incertain embodiments, the antisense compounds provided herein arespecifically hybridizable with a nrRNA.

Complementarity

An antisense compound and a target nucleic acid are complementary toeach other when a sufficient number of nucleobases of the antisensecompound can hydrogen bond with the corresponding nucleobases of thetarget nucleic acid, such that a desired effect will occur (e.g.,antisense inhibition of a target nucleic acid, such as a nrRNA).

An antisense compound can hybridize over one or more segments of a nrRNAsuch that intervening or adjacent segments are not involved in thehybridization event (e.g., a loop structure, mismatch or hairpinstructure).

In certain embodiments, the antisense compounds provided herein, or aspecified portion thereof, are, or are at least 70%, 80%, 85%, 86%, 87%,88, %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%complementary to a nrRNA, a target region, target segment, or specifiedportion thereof. In certain embodiments, the antisense compounds are atleast 70%, at least 80%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% complementary to a nrRNA, a targetregion, target segment, or specified portion thereof and contain atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, or atleast 19, contiguous nucleobases of the nucleobase sequence of any ofthe exemplary antisense compounds described herein. Percentcomplementarity of an antisense compound with a target nucleic acid canbe determined using routine methods.

For example, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases can be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden,Genome Res., 1997, 7, 649 656). Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, orspecified portions thereof, are fully complementary (i.e. 100%complementary) to a target nucleic acid, or specified portion thereof.For example, antisense compound can be fully complementary to targetnucleic acid, or a target region, or a target segment or target sequencethereof. As used herein, “fully complementary” means each nucleobase ofan antisense compound is capable of precise base pairing with thecorresponding nucleobases of a target nucleic acid. For example, a 20nucleobase antisense compound is fully complementary to a targetsequence that is 400 nucleobases long, so long as there is acorresponding 20 nucleobase portion of the target nucleic acid that isfully complementary to the antisense compound. Fully complementary canalso be used in reference to a specified portion of the first and/or thesecond nucleic acid. For example, a 20 nucleobase portion of a 30nucleobase antisense compound can be “fully complementary” to a targetsequence that is 400 nucleobases long. The 20 nucleobase portion of the30 nucleobase oligonucleotide is fully complementary to the targetsequence if the target sequence has a corresponding 20 nucleobaseportion wherein each nucleobase is complementary to the 20 nucleobaseportion of the antisense compound. At the same time, the entire 30nucleobase antisense compound can be fully complementary to the targetsequence, depending on whether the remaining 10 nucleobases of theantisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase can be at the 5′ end or3′ end of the antisense compound. Alternatively, the non-complementarynucleobase or nucleobases can be at an internal position of theantisense compound. When two or more non-complementary nucleobases arepresent, they can be either contiguous (i.e. linked) or non-contiguous.In one embodiment, a non-complementary nucleobase is located in the wingsegment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 10,12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas a nrRNA, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 10,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 nucleobases in length comprise no more than 6, no more than 5, nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas a nrRNA, or specified portion thereof.

The antisense compounds provided herein also include those which arecomplementary to a portion of a target nucleic acid. As used herein,“portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof an antisense compound. In certain embodiments, the antisensecompounds, are complementary to at least an 8 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds arecomplementary to at least a 10 nucleobase portion of a target segment.In certain embodiments, the antisense compounds are complementary to atleast a 15 nucleobase portion of a target segment. Also contemplated areantisense compounds that are complementary to at least an 8, at least a9, at least a 10, at least an 11, at least a 12, at least a 13, at leasta 14, at least a 15, at least a 16, at least a 17, at least a 18, atleast a 19, at least a 20, or more nucleobase portion of a targetsegment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein can also have a defined percentidentity to a particular nucleotide sequence, SEQ ID NO, or compoundrepresented by a specific Isis number, or portion thereof. As usedherein, an antisense compound is identical to the sequence disclosedherein if it has the same nucleobase pairing ability. For example, a RNAwhich contains uracil in place of thymidine in a disclosed DNA sequencewould be considered identical to the DNA sequence since both uracil andthymidine pair with adenine. Shortened and lengthened versions of theantisense compounds described herein as well as compounds havingnon-identical bases relative to the antisense compounds provided hereinalso are contemplated. The non-identical bases can be adjacent to eachother or dispersed throughout the antisense compound. Percent identityof an antisense compound is calculated according to the number of basesthat have identical base pairing relative to the sequence to which it isbeing compared.

In certain embodiments, the antisense compounds, or portions thereof,are at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%, identical to one or more of the antisense compounds or SEQID NOs, or a portion thereof, disclosed herein.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known asbase) portion of the nucleoside is normally a heterocyclic base moiety.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.Oligonucleotides are formed through the covalent linkage of adjacentnucleosides to one another, to form a linear polymeric oligonucleotide.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide.

Modifications to antisense compounds encompass substitutions or changesto internucleoside linkages, sugar moieties, or nucleobases. Modifiedantisense compounds are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for nucleic acid target, increased stability in thepresence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides can also be employed to increase thebinding affinity of a shortened or truncated antisense oligonucleotidefor its target nucleic acid. Consequently, comparable results can oftenbe obtained with shorter antisense compounds that have such chemicallymodified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5′ phosphodiester linkage. Antisense compounds having one or moremodified, i.e. non-naturally occurring, internucleoside linkages areoften selected over antisense compounds having naturally occurringinternucleoside linkages because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for target nucleicacids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom as well asinternucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates. Methods ofpreparation of phosphorus-containing and non-phosphorus-containinglinkages are well known.

In certain embodiments, antisense compounds targeted to a nrRNA compriseone or more modified internucleoside linkages. In certain embodiments,the modified internucleoside linkages are phosphorothioate linkages. Incertain embodiments, each internucleoside linkage of an antisensecompound is a phosphorothioate internucleoside linkage.

Modified Sugar Moieties

Antisense compounds of the invention can optionally contain one or morenucleosides wherein the sugar group has been modified. Such sugarmodified nucleosides may impart enhanced nuclease stability, increasedbinding affinity, or some other beneficial biological property to theantisense compounds. In certain embodiments, nucleosides comprise achemically modified ribofuranose ring moieties. Examples of chemicallymodified ribofuranose rings include without limitation, addition ofsubstitutent groups (including 5′ and 2′ substituent groups, bridging ofnon-geminal ring atoms to form bicyclic nucleic acids (BNA), replacementof the ribosyl ring oxygen atom with S, N(R), or C(R1)(R)2 (R═H, C1-C12alkyl or a protecting group) and combinations thereof. Examples ofchemically modified sugars include 2′-F-5′-methyl substituted nucleoside(see PCT International Application WO 2008/101157 Published on Aug. 21,2008 for other disclosed 5′,2′-bis substituted nucleosides) orreplacement of the ribosyl ring oxygen atom with S with furthersubstitution at the 2′-position (see published U.S. Patent ApplicationUS2005-0130923, published on Jun. 16, 2005) or alternatively5′-substitution of a BNA (see PCT International Application WO2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted withfor example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include withoutlimitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S,2′-F, 2′-OCH3 and 2′-O(CH2)2OCH3 substituent groups. The substituent atthe 2′ position can also be selected from allyl, amino, azido, thio,O-allyl, O—C1-C10 alkyl, OCF3, O(CH2)2SCH3, O(CH2)2-O—N(Rm)(Rn), andO—CH2-C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H orsubstituted or unsubstituted C1-C10 alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosidescomprising a bicyclic sugar moiety. Examples of bicyclic nucleosidesinclude without limitation nucleosides comprising a bridge between the4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisensecompounds provided herein include one or more bicyclic nucleosideswherein the bridge comprises a 4′ to 2′ bicyclic nucleoside. Examples ofsuch 4′ to 2′ bicyclic nucleosides, include but are not limited to oneof the formulae: 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′(ENA); 4′-CH(CH₃)—O-2′ and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof seeU.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′(and analogs thereof see published International ApplicationWO/2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ (and analogsthereof see published International Application WO/2008/150729,published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see published U.S. PatentApplication US2004-0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′,wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see U.S. Pat. No.7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (seeChattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′ (and analogs thereof see published InternationalApplication WO 2008/154401, published on Dec. 8, 2008). See, forexample: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63,10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379(Jul. 4, 2007); U.S. Pat. Nos. 7,053,207; 6,268,490; 6,770,748;6,794,499; 7,034,133; and 6,525,191; Elayadi et al., Curr. OpinionInvens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8,1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; andU.S. Pat. No. 6,670,461; International applications WO 2004/106356; WO94/14226; WO 2005/021570; U.S. Patent Publication Nos. US2004-0171570;US2007-0287831; US2008-0039618; U.S. Pat. No. 7,399,845; U.S. patentSer. No. 12/129,154; 60/989,574; 61/026,995; 61/026,998; 61/056,564;61/086,231; 61/097,787; 61/099,844; PCT International Applications Nos.PCT/US2008/064591; PCT/US2008/066154; PCT/US2008/068922; and PublishedPCT International Applications WO 2007/134181. Each of the foregoingbicyclic nucleosides can be prepared having one or more stereochemicalsugar configurations including for example α-L-ribofuranose andβ-D-ribofuranose (see PCT international application PCT/DK98/00393,published on Mar. 25, 1999 as WO 99/14226).

In certain embodiments, bicyclic sugar moieties of BNA nucleosidesinclude, but are not limited to, compounds having at least one bridgebetween the 4′ and the 2′ position of the pentofuranosyl sugar moietywherein such bridges independently comprises 1 or from 2 to 4 linkedgroups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl ora protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is,—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certainembodiments, the bridge is 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′,4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′-wherein each R is, independently, H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's havebeen incorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) BNA, (C) Ethyleneoxy (4′-(CH₂)₂—O2′) BNA, (D) Aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, and (F)Methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA, (G) methylene-thio(4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH2-N(R)-2′) BNA, (I) methylcarbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, and (J) propylene carbocyclic(4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is the base moiety and R is independently H, a protectinggroup or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleoside having Formula I:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—,—CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium.

In certain embodiments, bicyclic nucleoside having Formula II:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl,substituted acyl, substituted amide, thiol or substituted thio.

In one embodiment, each of the substituted groups is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃,OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) andJ_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl andX is O or NJ_(c).

In certain embodiments, bicyclic nucleoside having Formula III:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl orsubstituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleoside having Formula IV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl,substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl orsubstituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleoside having Formula V:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen,halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j),SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k),C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k),N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl.

The synthesis and preparation of the methyleneoxy (4′-CH₂—O-2′) BNAmonomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine anduracil, along with their oligomerization, and nucleic acid recognitionproperties have been described (Koshkin et al., Tetrahedron, 1998, 54,3607-3630). BNAs and preparation thereof are also described in WO98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) BNA, methyleneoxy (4′-CH₂—O-2′)BNA and 2′-thio-BNAs, have also been prepared (Kumar et al., Bioorg.Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleosideanalogs comprising oligodeoxyribonucleotide duplexes as substrates fornucleic acid polymerases has also been described (Wengel et al., WO99/14226). Furthermore, synthesis of 2′-amino-BNA, a novelcomformationally restricted high-affinity oligonucleotide analog hasbeen described in the art (Singh et al., J. Org. Chem., 1998, 63,10035-10039). In addition, 2′-Amino- and 2′-methylamino-BNA's have beenprepared and the thermal stability of their duplexes with complementaryRNA and DNA strands has been previously reported.

In certain embodiments, bicyclic nucleoside having Formula VI:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl,substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j),NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j),O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) orN(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(l) and q_(k) together are ═C(q_(g))(q_(h)),wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and thealkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Freier etal., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al.,J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation ofcarbocyclic bicyclic nucleosides along with their oligomerization andbiochemical studies have also been described (Srivastava et al., J. Am.Chem. Soc. 2007, 129(26), 8362-8379).

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclicnucleoside” refers to a bicyclic nucleoside comprising a furanose ringcomprising a bridge connecting two carbon atoms of the furanose ringconnects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

As used herein, “monocylic nucleosides” refer to nucleosides comprisingmodified sugar moieties that are not bicyclic sugar moieties. In certainembodiments, the sugar moiety, or sugar moiety analogue, of a nucleosidemay be modified or substituted at any position.

As used herein, “2′-modified sugar” means a furanosyl sugar modified atthe 2′ position. In certain embodiments, such modifications includesubstituents selected from: a halide, including, but not limited tosubstituted and unsubstituted alkoxy, substituted and unsubstitutedthioalkyl, substituted and unsubstituted amino alkyl, substituted andunsubstituted alkyl, substituted and unsubstituted allyl, andsubstituted and unsubstituted alkynyl. In certain embodiments, 2′modifications are selected from substituents including, but not limitedto: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂,OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m arefrom 1 to about 10. Other 2′-substituent groups can also be selectedfrom: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improvingpharmacokinetic properties, or a group for improving the pharmacodynamicproperties of an antisense compound, and other substituents havingsimilar properties. In certain embodiments, modified nucleosidescomprise a 2′-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272,11944-12000). Such 2′-MOE substitution have been described as havingimproved binding affinity compared to unmodified nucleosides and toother modified nucleosides, such as 2′-O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-MOE substituent also havebeen shown to be antisense inhibitors of gene expression with promisingfeatures for in vivo use (Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al.,Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., NucleosidesNucleotides, 1997, 16, 917-926).

As used herein, a “modified tetrahydropyran nucleoside” or “modified THPnucleoside” means a nucleoside having a six-membered tetrahydropyran“sugar” substituted in for the pentofuranosyl residue in normalnucleosides (a sugar surrogate). Modified THP nucleosides include, butare not limited to, what is referred to in the art as hexitol nucleicacid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA(F-HNA) or those compounds having Formula X:

Formula X:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the antisense compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl; and each of R₁ and R₂ is selectedfrom hydrogen, hydroxyl, halogen, substituted or unsubstituted alkoxy,NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein Xis O, S or NJ₁ and each J₁, J₂ and J₃ is, independently, H or C₁-C₆alkyl.

In certain embodiments, the modified THP nucleosides of Formula X areprovided wherein q_(m), q_(n), q_(p), q_(r), q_(s), q_(t) and q_(u) areeach H. In certain embodiments, at least one of q_(m), q_(n), q_(p),q_(r), q_(s), q_(t) and q_(u) is other than H. In certain embodiments,at least one of q_(m), q_(n), q_(p), q_(r), q_(s), q_(t) and q_(u) ismethyl. In certain embodiments, THP nucleosides of Formula X areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a sugar comprising a substituent at the 2′ position otherthan H or OH. 2′-modified nucleosides, include, but are not limited to,bicyclic nucleosides wherein the bridge connecting two carbon atoms ofthe sugar ring connects the 2′ carbon and another carbon of the sugarring; and nucleosides with non-bridging 2′substituents, such as allyl,amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.2′-modified nucleosides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugarcomprising a fluoro group at the 2′ position.

As used herein, “2′-OMe” or “2′-OCH₃” or “2′-O-methyl” each refers to anucleoside comprising a sugar comprising an —OCH₃ group at the 2′position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or“2′-O-methoxyethyl” each refers to a nucleoside comprising a sugarcomprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds (see for example review article:Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).Such ring systems can undergo various additional substitutions toenhance activity.

Methods for the preparations of modified sugars are well known to thoseskilled in the art.

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or morenucleotides having modified sugar moieties. In certain embodiments, themodified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOEmodified nucleotides are arranged in a gapmer motif. In certainembodiments, the modified sugar moiety is a cEt. In certain embodiments,the cEt modified nucleotides are arranged throughout the wings of agapmer motif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic unmodified nucleobases. Both natural and modifiednucleobases are capable of participating in hydrogen bonding. Suchnucleobase modifications can impart nuclease stability, binding affinityor some other beneficial biological property to antisense compounds.Modified nucleobases include synthetic and natural nucleobases such as,for example, 5-methylcytosine (5-me-C). Certain nucleobasesubstitutions, including 5-methylcytosine substitutions, areparticularly useful for increasing the binding affinity of an antisensecompound for a target nucleic acid. For example, 5-methylcytosinesubstitutions have been shown to increase nucleic acid duplex stabilityby 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds.,Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278).

Additional unmodified nucleobases include 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Heterocyclic base moieties can also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Nucleobases that are particularly useful for increasing the bindingaffinity of antisense compounds include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to a nrRNA compriseone or more modified nucleobases. In certain embodiments, gap-widenedantisense oligonucleotides targeted to a nrRNA comprise one or moremodified nucleobases. In certain embodiments, the modified nucleobase is5-methylcytosine. In certain embodiments, each cytosine is a5-methylcytosine.

Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense oligonucleotides can be admixed with pharmaceuticallyacceptable active or inert substance for the preparation ofpharmaceutical compositions or formulations. Compositions and methodsfor the formulation of pharmaceutical compositions are dependent upon anumber of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Antisense compound targeted to a nrRNA can be utilized in pharmaceuticalcompositions by combining the antisense compound with a suitablepharmaceutically acceptable diluent or carrier. A pharmaceuticallyacceptable diluent includes phosphate-buffered saline (PBS). PBS is adiluent suitable for use in compositions to be delivered parenterally.Accordingly, in one embodiment, employed in the methods described hereinis a pharmaceutical composition comprising an antisense compoundtargeted to a nrRNA and a pharmaceutically acceptable diluent. Incertain embodiments, the pharmaceutically acceptable diluent is PBS. Incertain embodiments, the antisense compound is an antisenseoligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other oligonucleotide which, upon administration to an animal,including a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure is also drawn to pharmaceutically acceptablesalts of antisense compounds, prodrugs, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents. Suitablepharmaceutically acceptable salts include, but are not limited to,sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an antisense compound which are cleaved by endogenousnucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds can be covalently linked to one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the resulting antisense oligonucleotides. Typical conjugategroups include cholesterol moieties and lipid moieties. Additionalconjugate groups include carbohydrates, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizinggroups that are generally attached to one or both termini of antisensecompounds to enhance properties such as, for example, nucleasestability. Included in stabilizing groups are cap structures. Theseterminal modifications protect the antisense compound having terminalnucleic acid from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be presenton both termini. Cap structures are well known in the art and include,for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizinggroups that can be used to cap one or both ends of an antisense compoundto impart nuclease stability include those disclosed in WO 03/004602published on Jan. 16, 2003.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level or activity of a nrRNAcan be tested in vitro in a variety of cell types. Cell types used forsuch analyses are available from commercial vendors (e.g. American TypeCulture Collection, Manassus, Va.; Zen-Bio, Inc., Research TrianglePark, N.C.; Clonetics Corporation, Walkersville, Md.) and cells arecultured according to the vendor's instructions using commerciallyavailable reagents (e.g. Invitrogen Life Technologies, Carlsbad,Calif.). Illustrative cell types include, but are not limited to, HepG2cells, Hep3B cells, primary hepatocytes, A549 cells, GM04281 fibroblastsand LLC-MK2 cells.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisenseoligonucleotides, which can be modified appropriately for treatment withother antisense compounds.

In general, cells are treated with antisense oligonucleotides when thecells reach approximately 60-80% confluence in culture.

One reagent commonly used to introduce antisense oligonucleotides intocultured cells includes the cationic lipid transfection reagentLIPOFECTIN® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotidesare mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, Calif.)to achieve the desired final concentration of antisense oligonucleotideand a LIPOFECTIN® concentration that typically ranges 2 to 12 ug/mL per100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes LIPOFECTAMINE 2000® (Invitrogen, Carlsbad,Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE 2000® inOPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) toachieve the desired concentration of antisense oligonucleotide and aLIPOFECTAMINE® concentration that typically ranges 2 to 12 ug/mL per 100nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes Cytofectin® (Invitrogen, Carlsbad, Calif.).Antisense oligonucleotide is mixed with Cytofectin® in OPTI-MEM® 1reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve thedesired concentration of antisense oligonucleotide and a Cytofectin®concentration that typically ranges 2 to 12 ug/mL per 100 nM antisenseoligonucleotide.

Another technique used to introduce antisense oligonucleotides intocultured cells includes electroporation.

Cells are treated with antisense oligonucleotides by routine methods.Cells are typically harvested 16-24 hours after antisenseoligonucleotide treatment, at which time RNA or protein levels of targetnucleic acids are measured by methods known in the art and describedherein. In general, when treatments are performed in multiplereplicates, the data are presented as the average of the replicatetreatments.

The concentration of antisense oligonucleotide used varies from cellline to cell line. Methods to determine the optimal antisenseoligonucleotide concentration for a particular cell line are well knownin the art. Antisense oligonucleotides are typically used atconcentrations ranging from 1 nM to 300 nM when transfected withLIPOFECTAMINE2000®, Lipofectin or Cytofectin. Antisense oligonucleotidesare used at higher concentrations ranging from 625 to 20,000 nM whentransfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are well known in the art. RNA is preparedusing methods well known in the art, for example, using the TRIZOL®Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer'srecommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or activity of a nrRNA can be assayed in a varietyof ways known in the art. For example, target nucleic acid levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or quantitative real-time PCR. RNA analysis can beperformed on total cellular RNA or poly(A)+ mRNA. Methods of RNAisolation are well known in the art. Northern blot analysis is alsoroutine in the art. Quantitative real-time PCR can be convenientlyaccomplished using the commercially available ABI PRISM® 7600, 7700, or7900 Sequence Detection System, available from PE-Applied Biosystems,Foster City, Calif. and used according to manufacturer's instructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels can be accomplished by quantitativereal-time PCR using the ABI PRISM® 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. Methods of quantitative real-time PCRare well known in the art.

Prior to real-time PCR, the isolated RNA is animaled to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents are obtained fromInvitrogen (Carlsbad, Calif.). RT, real-time-PCR reactions are carriedout by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR are normalizedusing either the expression level of a gene whose expression isconstant, such as cyclophilin A, or by quantifying total RNA usingRIBOGREEN® (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expressionis quantified by real time PCR, by being run simultaneously with thetarget, multiplexing, or separately. Total RNA is quantified usingRIBOGREEN® RNA quantification reagent (Invitrogen, Inc. Eugene, Oreg.).Methods of RNA quantification by RIBOGREEN® are taught in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR® 4000instrument (PE Applied Biosystems) is used to measure RIBOGREEN®fluorescence.

Probes and primers are designed to hybridize to a nrRNA sequence.Methods for designing real-time PCR probes and primers are well known inthe art, and can include the use of software such as PRIMER EXPRESS®Software (Applied Biosystems, Foster City, Calif.).

For Quantitation of nuclear structures, such as nuclear foci, speckles,paraspecles and other nuclear substructures, induced by ncRNA as measureof antisense effects, immunofluorescence can be used (see e.g, Example26). In situ hybridization can also be used to measure RNA.

Analysis of Protein Levels

Antisense inhibition of certain nrRNAs can be assessed by measuringassociated protein levels. Protein levels can be evaluated orquantitated in a variety of ways well known in the art, such asimmunoprecipitation, Western blot analysis (immunoblotting),enzyme-linked immunosorbent assay (ELISA), quantitative protein assays,protein activity assays (for example, caspase activity assays),immunohistochemistry, immunocytochemistry or fluorescence-activated cellsorting (FACS). Antibodies directed to a target can be identified andobtained from a variety of sources, such as the MSRS catalog ofantibodies (Aerie Corporation, Birmingham, Mich.), or can be preparedvia conventional monoclonal or polyclonal antibody generation methodswell known in the art.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are testedin animals to assess their ability to inhibit a nrRNA and producephenotypic changes. Testing can be performed in normal animals, or inexperimental disease models. For administration to animals, antisenseoligonucleotides are formulated in a pharmaceutically acceptablediluent, such as phosphate-buffered saline. Administration includesparenteral routes of administration. Following a period of treatmentwith antisense oligonucleotides, RNA is isolated from tissue and changesin nrRNA levels or activity are measured. In certain embodiments,changes in associated protein levels are also measured.

Certain Indications

In certain embodiments, provided herein are methods of treating anindividual comprising administering one or more pharmaceuticalcompositions as described herein. In certain embodiments, the individualhas a disease or condition associated with a nuclear-retained RNA.

Accordingly, provided herein are methods for ameliorating a symptomassociated with a disease or condition associated with anuclear-retained RNA in an animal in need thereof. In certainembodiments, provided is a method for reducing the rate of onset of asymptom associated with a disease or condition associated with anuclear-retained RNA. In certain embodiments, provided is a method forreducing the severity of a symptom associated with a disease orcondition associated with a nuclear-retained RNA. In such embodiments,the methods comprise administering to an individual in need thereof atherapeutically effective amount of a compound targeted to a nrRNA.

In certain embodiments, administration of an antisense compound targetedto a nrRNA results in reduction of nrRNA levels by at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95% or at least about 99%, or a rangedefined by any two of these values.

In certain embodiments, pharmaceutical compositions comprising anantisense compound targeted to a nrRNA are used for the preparation of amedicament for treating a patient suffering or susceptible to a diseaseor condition associated with a nuclear-retained RNA.

Administration

In certain embodiments, the compounds and compositions as describedherein are administered parenterally.

In certain embodiments, parenteral administration is by injection. Theinjection can be delivered with a syringe.

Certain Combination Therapies

In certain embodiments, a first agent comprising the modifiedoligonucleotide of the invention is co-administered with one or moresecondary agents. In certain embodiments, such second agents aredesigned to treat the same a disease or condition associated with anuclear-retained RNA as the first agent described herein. In certainembodiments, such second agents are designed to treat a differentdisease, disorder, or condition as the first agent described herein. Incertain embodiments, such second agents are designed to treat anundesired side effect of one or more pharmaceutical compositions asdescribed herein. In certain embodiments, second agents areco-administered with the first agent to treat an undesired effect of thefirst agent. In certain embodiments, second agents are co-administeredwith the first agent to produce a combinational effect. In certainembodiments, second agents are co-administered with the first agent toproduce a synergistic effect.

In certain embodiments, a first agent and one or more second agents areadministered at the same time. In certain embodiments, the first agentand one or more second agents are administered at different times. Incertain embodiments, the first agent and one or more second agents areprepared together in a single pharmaceutical formulation. In certainembodiments, the first agent and one or more second agents are preparedseparately.

EXAMPLES NON-LIMITING DISCLOSURE AND INCORPORATION BY REFERENCE

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1 Antisense Inhibition of Human MALAT1 in A549 Cells

Antisense oligonucleotides targeted to a metastasis-associated lungadenocarcinoma transcript 1 (MALAT1) nucleic acid, a non-codingnuclear-retained RNA transcript, were tested for their effect on MALAT1RNA transcript in vitro. Cultured A549 cells at a density of 5,000 cellsper well were transfected using Lipofectin reagent with 60 nM antisenseoligonucleotide. After approximately 24 hours, RNA was isolated from thecells and MALAT1 RNA transcript levels were measured by quantitativereal-time PCR. The human primer probe set RTS2736 (forward sequenceAAAGCAAGGTCTCCCCACAAG, designated herein as SEQ ID NO: 89; reversesequence TGAAGGGTCTGTGCTAGATCAAAA, designated herein as SEQ ID NO: 90;probe sequence TGCCACATCGCCACCCCGTX, designated herein as SEQ ID NO: 91)was used to quantitated MALAT1 RNA. MALAT1 RNA transcript levels wereadjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of MALAT1, relative tountreated control cells.

The antisense oligonucleotides in Table 3 are 5-10-5 gapmers, where thegap segment comprises ten 2′-deoxynucleosides and each wing segmentcomprises five 2′-MOE nucleosides. Each nucleotide in the 5′ wingsegment and each nucleotide in the 3′ wing segment has a 2′-MOEmodification. The internucleoside linkages throughout each gapmer arephosphorothioate (P═S) linkages. All cytidine residues throughout eachgapmer are 5-methylcytidines. ‘Target start site’ indicates the 5′-mostnucleotide to which the antisense oligonucleotide is targeted. ‘Targetstop site’ indicates the 3′-most nucleotide to which the antisenseoligonucleotide is targeted. All the antisense oligonucleotides listedin Table 3 target SEQ ID NO: 1 (GENBANK Accession No. EF177381).

TABLE 3 Inhibition of human MALAT1 RNA transcript in A549cells by 5-10-5 gapmers targeting SEQ ID NO: 1 Target Target % SEQ StartStop ISIS inhibi- ID Site Site No. Sequence (5′ to 3′) tion NO 3520 3539395240 TGCCTTTAGGATTCTAGACA 88 92 4085 4104 395243 TAATTGCCAATATTTGCCCC82 93 4236 4255 395244 GGGAGTTACTTGCCAACTTG 89 94 4692 4711 395248TTGCAGTTAAACAATGGAAA 84 95 4897 4916 395251 CCAGGCTGGTTATGACTCAG 89 964947 4966 395252 TTATCAATTCACCAAGGAGC 88 97 4982 5001 395253ATGGAGGTATGACATATAAT 90 98 5042 5061 395254 GGCATATGCAGATAATGTTC 87 995322 5341 395255 ACATTGGCACACAGCACAGC 86 100 5333 5352 395256AGGCAAACGAAACATTGGCA 90 101 5447 5466 395257 CTAACATGCAATACTGCAGA 88 1025592 5611 395259 AAGCCCACAGGAACAAGTCC 84 103 6297 6316 395267GGTCAATAGTGTAAAACATT 87 104 6373 6392 395269 TTCATGAAGGATGAAATGCC 84 1056644 6663 395272 CAATGCATTCTAATAGCAGC 85 106 6958 6977 395275AACATTTCCACTTGCCAGTT 87 107 7157 7176 395280 GGTTCCCAATCCCCACATTT 89 1087534 7553 395283 TAATAAAAATCAGGTGAGGC 85 109 8077 8096 395287TCCCACCCAGCATTACAGTT 84 110

Example 2 Antisense Inhibition of Human MALAT1 in A549 Cells

Antisense oligonucleotides targeted to a MALAT1 nucleic acid were testedfor their effects on MALAT1 RNA in vitro. Cultured A549 cells at adensity of 5,000 cells per well were transfected using Lipofectinreagent with 150 nM antisense oligonucleotide. After approximately 24hours, RNA was isolated from the cells and MALAT1 RNA transcript levelswere measured by quantitative real-time PCR. Human primer probe setRTS2736 was used to quantitate MALAT1 RNA. MALAT1 RNA transcript levelswere adjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of MALAT1, relative tountreated control cells.

The antisense oligonucleotides in Table 4 are 5-10-5 gapmers, where thegap segment comprises ten 2′-deoxynucleosides and each wing segmentcomprises five 2′-MOE nucleosides. Each nucleotide in the 5′ wingsegment and each nucleotide in the 3′ wing segment has a 2′-MOEmodification. The internucleoside linkages throughout each gapmer arephosphorothioate (P═S) linkages. All cytidine residues throughout eachgapmer are 5-methylcytidines. ‘Target start site’ indicates the 5′-mostnucleotide to which the antisense oligonucleotide is targeted. ‘Targetstop site’ indicates the 3′-most nucleotide to which the antisenseoligonucleotide is targeted. All the antisense oligonucleotides listedin Table 4 target SEQ ID NO: 1 (GENBANK Accession No. EF177381).

TABLE 4 Inhibition of human MALAT1 RNA transcript in A549cells by 5-10-5 gapmers targeting SEQ ID NO: 1 Target Target % SEQ StartStop ISIS inhibi- ID Site Site No. Sequence (5′ to 3′) tion NO 3520 3539395240 TGCCTTTAGGATTCTAGACA 87 92 4085 4104 395243 TAATTGCCAATATTTGCCCC88 93 4236 4255 395244 GGGAGTTACTTGCCAACTTG 88 94 4692 4711 395248TTGCAGTTAAACAATGGAAA 85 95 4897 4916 395251 CCAGGCTGGTTATGACTCAG 85 964947 4966 395252 TTATCAATTCACCAAGGAGC 92 97 4982 5001 395253ATGGAGGTATGACATATAAT 93 98 5042 5061 395254 GGCATATGCAGATAATGTTC 75 995322 5341 395255 ACATTGGCACACAGCACAGC 86 100 5333 5352 395256AGGCAAACGAAACATTGGCA 95 101 5447 5466 395257 CTAACATGCAATACTGCAGA 81 1025592 5611 395259 AAGCCCACAGGAACAAGTCC 86 103 6297 6316 395267GGTCAATAGTGTAAAACATT 84 104 6373 6392 395269 TTCATGAAGGATGAAATGCC 86 1056644 6663 395272 CAATGCATTCTAATAGCAGC 90 106 6958 6977 395275AACATTTCCACTTGCCAGTT 85 107 7157 7176 395280 GGTTCCCAATCCCCACATTT 86 1087534 7553 395283 TAATAAAAATCAGGTGAGGC 84 109 8077 8096 395287TCCCACCCAGCATTACAGTT 91 110

Example 3 Dose-Dependent Antisense Inhibition of Human MALAT1 in A549Cells

Several of the antisense oligonucleotides exhibiting in vitro inhibitionof MALAT1 in A549 cells (see Example 2) were tested at various doses.Cells were plated at a density of 5,000 cells per well and transfectedusing Lipofectin reagent with 7.5 nM, 15 nM, 30 nM, and 60 nMconcentrations of each antisense oligonucleotide. After approximately 16hours, RNA was isolated from the cells and MALAT1 RNA transcript levelswere measured by quantitative real-time PCR using primer probe setRTS2736 MALAT1 RNA transcript levels were normalized to total RNAcontent, as measured by RIBOGREEN®. Results are presented in Table 5 aspercent inhibition of MALAT1, relative to untreated control cells.

TABLE 5 Dose-dependent antisense inhibition of human MALAT1 in A549cells tested with primer probe set RTS2736 ISIS IC₅₀ No. 7.5 nM 15 nM 30nM 60 nM (nM) 395240 67 81 90 96 1.8 395243 31 54 80 93 13.1 395244 7673 86 91 0.5 395248 43 70 87 93 8.1 395251 57 73 84 90 4.0 395252 48 6988 91 6.9 395253 54 73 90 93 5.1 395254 67 74 82 75 0.1 395255 39 69 8292 9.4 395256 58 81 88 92 3.4 395257 60 78 89 93 3.3 395259 26 59 74 9114.1 395267 36 64 79 88 10.6 395269 36 63 82 92 10.7 395272 55 65 82 916.0 395275 57 76. 88 88 3.6 395280 68 82 87 86 0.6 395283 31 65 80 9011.8 395287 21 74 78 87 12.7

To test whether the inhibition of MALAT1 by the various oligonucleotideswas uniform across the MALAT1 transcript, the above dose-responseexperiment was repeated with two additional primer probe sets.

Probe set RTS 2738 has forward sequence of GAATTGCGTCATTTAAAGCCTAGTT,SEQ ID NO: 111; reverse sequence of TCATCCTACCACTCCCAATTAATCT, SEQ IDNO: 112; and a probe sequence of ACGCATTTACTAAACGCAGACGAAAATGGAX, SEQ IDNO: 113.

Probe set RTS 2739 has a forward sequence of AGGCGTTGTGCGTAGAGGAT, SEQID NO: 114; a reverse sequence of AAAGGTTACCATAAGTAAGTTCCAGAAAA, SEQ IDNO: 115; and a probe sequence of AGTGGTTGGTAAAAATCCGTGAGGTCGGX, SEQ IDNO: 116.

Results are presented in Tables 6 and 7 as percent inhibition of MALAT1,relative to untreated control cells.

TABLE 6 Dose-dependent antisense inhibition of human MALAT1 in A549cells, primer probe set RTS2738 ISIS IC₅₀ No. 7.5 nM 15 nM 30 nM 60 nM(nM) 395240 67 79 87 90 1.4 395243 29 55 76 90 13.9 395244 57 70 81 904.7 395248 35 64 84 89 10.6 395251 62 71 79 88 2.8 395252 46 66 84 897.6 395253 49 72 87 92 6.2 395254 61 75 79 84 2.0 395255 31 63 79 8812.1 395256 55 78 85 89 4.0 395257 56 73 86 90 4.4 395259 18 58 75 8615.9 395267 30 60 77 87 12.8 395269 28 53 81 90 13.9 395272 45 57 76 879.7 395275 52 71 83 85 5.2 395280 60 76 83 88 2.6 395283 23 51 72 8416.4 395287 10 65 72 84 16.8

TABLE 7 Dose-dependent antisense inhibition of human MALAT1 in A549cells, primer probe set RTS2739 ISIS IC₅₀ No. 7.5 nM 15 nM 30 nM 60 nM(nM) 395240 69 81 89 93 1.2 395243 28 77 91 13.5 395244 59 72 85 91 4.0395248 42 64 86 91 8.9 395251 65 74 83 91 2.3 395252 51 72 87 91 5.8395253 51 73 90 94 5.8 395254 66 74 82 88 1.6 395255 39 64 84 90 9.8395256 58 80 88 92 3.3 395257 58 75 91 93 4.0 395259 22 58 75 88 15.1395267 43 64 78 90 9.1 395269 34 56 79 92 12.4 395272 50 60 78 90 8.0395275 55 76 87 87 3.8 395280 60 80 85 91 2.8 395283 32 54 74 84 13.9395287 16 73 73 86 14.4

Example 4 Dose-Dependent Antisense Inhibition of Human MALAT1 in HeLaCells

Several antisense oligonucleotides exhibiting in vitro inhibition ofMALAT1 were tested at various doses in HeLa cells. Cells were plated ata density of 5,000 cells per well and transfected using Lipofectinreagent with 4.7 nM, 9.4 nM, 18.8 nM, 37.8 nM, 75 nM, and 150 nMconcentrations of each antisense oligonucleotide, as indicated in Tables8 and 9. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and MALAT1 RNA transcript levels were measuredby quantitative real-time PCR. Human MALAT1 primer probe set RTS2736 andRTS2738 (see Example 3) were used to measure RNA transcript levels.MALAT1 RNA transcript levels were adjusted according to total RNAcontent, as measured by RIBOGREEN®. Results are presented as percentinhibition of MALAT1, relative to untreated control cells. Asillustrated in Tables 8 and 9, MALAT1 RNA transcript levels were reducedin a dose-dependent manner.

TABLE 8 Dose-dependent antisense inhibition of human MALAT1 in HeLa cellline, primer probe set RTS2736 ISIS 4.7 9.4 18.8 37.8 75 150 IC₅₀ No nMnM nM nM nM nM (nM) 395240 25 36 54 68 85 95 16.2 395254 0 25 26 46 6475 44.1 395280 6 2 41 63 84 91 28.7

TABLE 9 Dose-dependent antisense inhibition of human MALAT1 in HeLa cellline, primer probe set RTS2738 ISIS 4.7 9.4 18.8 37.8 75 150 IC₅₀ No. nMnM nM nM nM nM (nM) 395240 34 45 60 68 83 95 11.9 395254 0 18 19 50 7693 34.7 395280 8 3 32 56 79 90 32.3

Example 5 Dose-Dependent Antisense Inhibition of Human MALAT1 in HeLaCells

Several antisense oligonucleotides exhibiting in vitro inhibition ofMALAT1 were tested at various doses in HeLa cells. Cells were plated ata density of 4,000 cells per well and transfected using Lipofectinreagent with 3.7 nM, 11.1 nM, 33.3 nM, and 100.0 nM concentrations ofeach antisense oligonucleotide. After a treatment period of 16 hours,RNA was isolated from the cells and MALAT1 RNA transcript levels weremeasured by quantitative real-time PCR. MALAT1 RNA transcript levelswere adjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of MALAT1, relative tountreated control cells. As illustrated in Table 10, MALAT1 RNAtranscript levels were reduced in a dose-dependent manner.

TABLE 10 Dose-dependent antisense inhibition of human MALAT1 in HeLacells ISIS IC₅₀ No. 3.7 nM 11.1 nM 33.3 nM 100 nM (nM) 395240 68 87 9598 0.3 395280 59 86 97 97 0.9 395254 68 89 88 86 0.02 395244 57 84 97 981.2 395256 56 85 97 97 1.2 395275 34 64 91 98 6.5

Example 6 Dose-Dependent Antisense Inhibition of Human MALAT1 in U87MGCells

Several antisense oligonucleotides exhibiting in vitro inhibition ofMALAT1 were tested at various doses in U87MG cells. Cells were plated ata density of 4,000 cells per well and transfected using Lipofectinreagent with 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM concentrations of eachantisense oligonucleotide. After a treatment period of 16 hours, RNA wasisolated from the cells and MALAT1 RNA transcript levels were measuredby quantitative real-time PCR. MALAT1 RNA transcript levels wereadjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of MALAT1, relative tountreated control cells. As illustrated in Table 11, MALAT1 RNAtranscript levels were reduced in a dose-dependent manner.

TABLE 11 Dose-dependent antisense inhibition of human MALAT1 in U87MGcells ISIS IC₅₀ No. 3.7 nM 11.1 nM 33.3 nM 100 nM (nM) 395240 24 52 7490 11.7 395280 25 48 77 83 12.3 395254 9 39 67 71 23.0 395244 18 45 7887 14.0 395256 23 45 83 86 12.3 395275 3 45 73 89 17.5

Example 7 Dose-Dependent Antisense Inhibition of Human MALAT1 in HeLaCells by siRNAs

Several siRNAs specific for human MALAT1 RNA transcript were tested atvarious doses in the HeLa cell line. Cells were plated at a density of5,000 cells per well and transfected using LipofectAMINE2000® reagentwith 0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nMconcentrations of each siRNA. After incubation for approximately 4hours, transfection media was discarded, fresh media added, and thecells were further incubated for 18 hours. RNA was isolated from thecells and MALAT1 RNA transcript levels were measured by quantitativereal-time PCR. Primer probe set RTS2739 was used to measure RNAtranscript levels. Table 12 presents the results of inhibition comparedto untreated control cells.

TABLE 12 siRNA inhibition of human MALAT1 RNA transcript compared tocontrol 0.78 1.56 3.13 6.25 12.5 25 50 100 IC₅₀ nM nM nM nM nM nM nM nM(nM) MALAT-1 38 14 0 19 40 44 44 55 109.8 MALAT-2 18 21 45 51 62 73 7670 n.d. MALAT-3 0 0 0 9 23 17 25 22 n.d. MALAT-4 0 8 10 19 40 38 49 39103.4 n.d. = no data

Example 8 Dose Response Studies with Antisense Oligonucleotides andCorrelation with RNA Stability in b.END Cells

The correlation of non-coding RNA stability of the murinenuclear-retained target molecule, MALAT1, to the dose of antisenseoligonucleotide required to achieve target knockdown was studied.

Antisense oligonucleotides, ISIS 395251 (CCAGGCTGGTTATGACTCAG; targetsite 3338), incorporated herein as SEQ ID NO: 96; ISIS 399462(GGGTCAGCTGCCAATGCTAG, target site 1280), incorporated herein as SEQ IDNO: 117; ISIS 399479 (CGGTGCAAGGCTTAGGAATT, target site 4004),incorporated herein as SEQ ID NO: 118; ISIS 399488(TTACCCTAGATGTTTAGCCA, target site 4621), incorporated herein as SEQ IDNO: 119; ISIS 399495 (GAAAATGGCATGTCTGCTTC, target site 120),incorporated herein as SEQ ID NO: 120; ISIS 399462(GGGTCAGCTGCCAATGCTAG, target site 1280), incorporated herein as SEQ IDNO: 117; ISIS 395290 (TAAGATGCTAGCTTGGCCAA, target site 6552),incorporated herein as SEQ ID NO: 121; ISIS 395275(AACATTTCCACTTGCCAGTT, target site 5348), incorporated herein as SEQ IDNO: 107; ISIS 399503 (AAATTGATGGCCTTTTCTGG, target site 6316),incorporated herein as SEQ ID NO: 122; ISIS 399473(ATATGCAGCTTTTCATCAGT, target site 3475), incorporated herein as SEQ IDNO: 123; and ISIS 399484 (ACAAGTACATTGGAGCACAT, target site 4206),incorporated herein as SEQ ID NO: 124; all targeting murine MALAT1 RNAtranscript (GENBANK Accession No. 3144_(—)097A, designated herein as SEQID NO: 125) were tested at various doses. b.END cells were plated at adensity of 4,000 cells per well and transfected using Cytofectin reagentwith 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM concentrationsof each antisense oligonucleotide. After approximately 16 hours, RNA wasisolated from the cells and MALAT-1 RNA transcript levels were measuredby quantitative real-time PCR using primer probe set mMALAT1#2 (forwardsequence TGGGTTAGAGAAGGCGTGTACTG, designated herein as SEQ ID NO: 126;reverse sequence TCAGCGGCAACTGGGAAA, designated herein as SEQ ID NO:127; and probe sequence CGTTGGCACGACACCTTCAGGGACTX, designated herein asSEQ ID NO: 128). MALAT1 RNA transcript levels were normalized to totalRNA content, as measured by RIBOGREEN®. Results are presented in Table13 as percent inhibition of MALAT1, relative to untreated control cells.

All the gapmers of Table 13 are 5-10-5 gapmers, where the gap segmentcomprises ten 2′-deoxynucleosides and each wing segment comprises five2′-MOE nucleosides. The internucleoside linkages throughout each gapmerare phosphorothioate (P═S) linkages. All cytidine residues throughouteach gapmer are 5-methylcytidines.

To measure the stability of the MALAT1 non-coding RNA, b.END cells weretreated with 8 μg/mL of actinomycin D over a period of 10 hours. Anotherset of cells were treated with 75 μM5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) for the same timeperiod. Both RNA synthesis inhibitors gave similar results, as presentedin Table 14. Table 14 presents the percent mRNA levels compared to thePBS control after treatment with actinomycin D or DRB at different timepoints. The data demonstrates that the MALAT1 RNA does not get degradedand is therefore stable for the period of treatment.

The data from Tables 13 and 14 demonstrates that the IC₅₀ foroligonucleotides targeting RNA transcripts which are stable is low,suggesting that these RNA transcripts are more amenable for targetingcompared to RNA transcripts which are not as stable. Therefore, theseantisense oligonucleotides will be useful to employ in cases whereoligonucleotide uptake by cells is low.

TABLE 13 Dose-dependent antisense inhibition of nuclear-retained RNA inb.END cells ISIS 3.125 6.25 12.5 25.0 50.0 100.0 IC₅₀ No. nM nM nM nM nMnM (nM) 399479 14 31 55 71 84 91 12.6 399484 10 35 48 69 86 91 13.5399488 10 11 32 52 76 85 22.3 399495 10 35 46 65 83 90 14.5 399462 20 3650 68 81 92 12.3 395290 16 31 48 66 84 89 13.7 395251 23 45 57 66 85 9010.1 395275 22 30 46 57 76 86 15.2 399503 21 28 31 52 78 81 19.1 39947329 32 45 57 79 86 13.5

TABLE 14 RNA levels (% PBS control) of nuclear-retained RNA at varioustime-points after treatment with actinomycin D or DRB Time (hrs)actinomycin DRB 0.00 108 125 0.25 124 106 0.50 161 126 0.75 160 107 1.00147 131 2.00 136 138 3.00 163 133 4.00 151 139 5.00 167 134 6.00 137 1317.00 135 105 8.00 155 116 9.00 143 113 10.00 131 118

Example 9 Dose Response Studies with Antisense Oligonucleotides andCorrelation with Murine Target mRNA Stability in b.END Cells

The correlation of mRNA stability of the murine target molecule to thedose of antisense oligonucleotide required to achieve target knockdownwas studied.

Antisense oligonucleotides, ISIS 5, ISIS 6, ISIS 7, ISIS 8, ISIS 9, ISIS10, ISIS 11, ISIS 12, ISIS 13, and ISIS 14 targeting ‘Target 3’ genesequence were tested at various doses. b.END cells were plated at adensity of 4,000 cells per well and transfected using Cytofectin reagentwith 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM concentrationsof each antisense oligonucleotide. After approximately 16 hours, RNA wasisolated from the cells and ‘Target 3’ RNA transcript levels weremeasured by quantitative real-time PCR. ‘Target 3’ RNA transcript levelswere normalized to total RNA content, as measured by RIBOGREEN®. Resultsare presented in Table 15 as percent inhibition of ‘Target 3’, relativeto untreated control cells.

Antisense oligonucleotides, ISIS F, ISIS G, ISIS H , ISIS I, ISIS J,ISIS K, ISIS L, ISIS M, ISIS N, and ISIS O targeting ‘Target 4’ mRNAwere tested at various doses. b.END cells were plated at a density of4,000 cells per well and transfected using Cytofectin reagent with1.4063 nM, 2.8125 nM, 5.625 nM, 11.25 nM, 22.5 nM and 45 nMconcentrations of each antisense oligonucleotide. After approximately 16hours, RNA was isolated from the cells and ‘Target 4’ RNA transcriptlevels were measured by quantitative real-time PCR. ‘Target 4’ RNAtranscript levels were normalized to total RNA content, as measured byRIBOGREEN®. Results are presented in Table 16 as percent inhibition of‘Target 4’, relative to untreated control cells.

All the gapmers of Tables 15 and 16 are 5-10-5 gapmers, where the gapsegment comprises ten 2′-deoxynucleosides and each wing segmentcomprises five 2′-MOE nucleosides. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P═S) linkages. All cytidineresidues throughout each gapmer are 5-methylcytidines.

The data presented in Tables 15 and 16 demonstrate that the IC₅₀ forantisense oligonucleotides targeting ‘Target 4’ is about twenty timeslower than the IC₅₀ for antisense oligonucleotides targeting ‘Target 3’in the same cell line and for gapmers with similar motif.

To measure the stability of the target mRNAs, b.END cells were treatedwith 8 μg/mL of actinomycin D over a period of 9 hours. Another set ofcells were treated with 75 μM5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) for the same timeperiod. Both RNA synthesis inhibitors gave similar results, as presentedin Tables 17 and 18. Tables 17 and 18 present the percent mRNA levelscompared to the PBS control after treatment with actinomycin D or DRB atdifferent time points. The data demonstrates that ‘Target 3’ mRNA hassignificantly less stability than ‘Target 4’ mRNA.

The data from Tables 15-18 suggests that antisense inhibition of targetmRNAs, which are comparatively more stable, is easier to achieve thanantisense inhibition of target mRNAs with less stability.

TABLE 15 Dose-dependent antisense inhibition of ‘Target 3’ mRNA in b.ENDcells ISIS 5 ISIS 6 ISIS 7 ISIS 8 ISIS 9 ISIS 10 ISIS 11 ISIS 12 ISIS 13ISIS 14 6.25 nM 13 18 25 24 0 0 15 2 18 21 12.5 nM 31 24 8 8 0 19 21 2222 27   25 nM 24 32 12 18 6 28 25 4 30 33   50 nM 43 43 39 24 40 43 5336 58 42  100 nM 40 82 53 39 74 51 86 62 n.d. n.d.  200 nM 57 71 24 73n.d. 49 n.d. n.d. n.d. 54 IC₅₀ 147 46 1239 134 62 121 38 91 47 115 (nM)n.d. = no data

TABLE 16 Dose-dependent antisense inhibition of ‘Target 4’ mRNA in b.ENDcells ISIS F ISIS G ISIS H ISIS I ISIS J ISIS K ISIS L ISIS M ISIS NISIS O 1.4063 5 20 25 0 26 24 25 26 18 21 nM 2.8125 32 37 32 21 46 46 4336 28 17 nM 5.625 67 56 61 50 68 69 60 62 50 34 nM 11.25 91 87 87 82 8892 92 78 77 69 nM 22.5 98 96 96 92 93 98 98 94 90 86 nM 45.0 98 99 96 9093 97 95 93 94 89 nM IC₅₀ 4.6 4.2 4 6.6 3.1 3.1 3.4 4 5.4 7.1 (nM)

TABLE 17 mRNA levels (% PBS control) of ‘Target 3’ at varioustime-points after treatment with actinomycin D or DRB Time (hrs)actinomycin D DRB 0 100 100 0.15 81 97 0.3 73 62 0.45 77 39 1 38 41 1.1528 18 1.3 20 13 1.45 11 7 2.45 4 5 3.45 2 11 4.45 3 15 5.45 2 31 6.45 236 7.45 2 44 8.45 3 50

TABLE 18 mRNA levels (% PBS control) of ‘Target 4’ at varioustime-points after treatment with actinomycin D or DRB Time (hrs)actinomycin DRB 0.00 100 100 0.25 91 116 0.5 90 109 0.75 86 67 1.00 88103 1.25 88 105 1.50 99 114 1.75 78 93 2.75 79 101 3.75 66 71 4.75 72 645.75 56 51 6.75 52 48 7.75 47 44 8.75 53 54

SR-B1 Stability

To measure the stability of the SR-B1 mRNA, b.END cells were treatedwith 8 μg/mL of actinomycin D over a period of 10 hours. Another set ofcells were treated with 75 μM5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) for the same timeperiod. Both RNA synthesis inhibitors gave similar results, as presentedin Table 19. Table 19 presents the percent mRNA levels compared to thePBS control after treatment with actinomycin D or DRB at different timepoints. The data demonstrates that SR-B1 mRNA does not get significantlydegraded upto 10 hours of the treatment and is therefore stable for theperiod of treatment.

TABLE 19 RNA levels (% PBS control) of SR-B1 mRNA at various time-pointsafter treatment with actinomycin D or DRB Time (hrs) actinomycin DRB0.25 90 125 0.50 91 110 0.75 89 78 1.00 87 116 1.75 81 119 2.00 89 1353.00 84 79 4.00 74 89 5.00 82 94 6.00 72 90 7.00 67 77 8.00 71 68 9.0066 74 10.00 63 61

Example 10 Dose Response Studies with Antisense Oligonucleotides andCorrelation with Target mRNA Stability in HUVEC Cells

The correlation of mRNA stability of the human target molecule to thedose of antisense oligonucleotide required to achieve target knockdownwas studied.

Antisense oligonucleotides, ISIS 1, ISIS 2, ISIS 3, and ISIS 4,targeting ‘Target 1’ mRNA were tested at various doses. HUVEC cells wereplated at a density of 5,000 cells per well and transfected usingLipoectAMINE2000® reagent with 0.0069 nM, 0.0206 nM, 0.0617 nM, 0.1852nM, 0.5556 nM, 1.6667 nM, 5 nM and 15 nM concentrations of eachantisense oligonucleotide. After approximately 16 hours, RNA wasisolated from the cells and ‘Target 1’ RNA transcript levels weremeasured by quantitative real-time PCR. ‘Target 1’ RNA transcript levelswere normalized to total RNA content, as measured by RIBOGREEN®. Resultsare presented in Table 20 as percent inhibition of ‘Target 1’, relativeto untreated control cells.

Antisense oligonucleotides, ISISA, ISIS B, ISIS C, ISIS D, and ISIS Etargeting ‘Target 2’ mRNA were tested at various doses. HUVEC cells wereplated at a density of 5,000 cells per well and transfected usingLipoectAMINE2000® reagent with 0.6173 nM, 1.8519 nM, 5:5556 nM, 16.6667nM, 50 nM and 150 nM concentrations of each antisense oligonucleotide.After approximately 16 hours, RNA was isolated from the cells and‘Target 2’ RNA transcript levels were measured by quantitative real-timePCR.

‘Target 2’ RNA transcript levels were normalized to total RNA content,as measured by RIBOGREEN®. Results are presented in Table 21 as percentinhibition of ‘Target 2’, relative to untreated control cells.

All the gapmers of Tables 20 and 21 are 2-13-5 gapmers, where the gapsegment comprises thirteen 2′-deoxynucleosides and the 5′ wing segmentcomprises two 2′-MOE nucleosides and the 3′ wing segment comprises five2′-MOE nucleosides. The internucleoside linkages throughout each gapmerare phosphorothioate (P═S) linkages. All cytidine residues throughouteach gapmer are 5-methylcytidines.

The data presented in Tables 20 and 21 demonstrate that the IC₅₀ forantisense oligonucleotides targeting ‘Target 1’ is ten times lower thanthe IC₅₀ for antisense oligonucleotides targeting ‘Target 2’ in the samecell line and for gapmers with similar motif.

To measure the stability of the target mRNAs, HUVEC cells were treatedwith 8 μg/mL of actinomycin D over a period of 9 hours. Another set ofcells were treated with 75 μM5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) for the same timeperiod. Another set of cells were 30 μg/mL of α-amanitin for the sametime period. At time points indicated in Tables 21 and 22, mRNA washarvested and ‘Target 1’ or ‘Target 2’ mRNA levels were quantified byRT-PCR. All three RNA synthesis inhibitors gave similar results, aspresented in Tables 22 and 23. Table 22 presents the percent mRNA levelscompared to the PBS control after treatment with actinomycin D or DRB atdifferent time points. Table 23 presents the percent mRNA levelscompared to the PBS control after treatment with DRB and α-amanitin atdifferent time points. Based on the results from the two differentassays, the half-life of ‘Target 2’ mRNA is calculated to be within 2-4hours. The half-life of ‘Target 1’ mRNA could not be calculated with theavailable data and is therefore beyond 9 hours. Therefore, the data inTables 22 and 23 demonstrates that the ‘Target 2’ mRNA has significantlyless stability than ‘Target 1’ mRNA.

The data from Tables 20-23 suggests that antisense inhibition of targetmRNAs, which are comparatively more stable, is easier to achieve thanantisense inhibition of target mRNAs with less stability.

TABLE 20 Dose-dependent antisense inhibition of ‘Target 1’ mRNA in HUVECcells ISIS 1 ISIS 2 ISIS 3 ISIS 4 0.0069 nM 0 9 5 19 0.0206 nM 14 26 2645 0.0617 nM 31 45 41 58 0.1852 nM 56 59 58 76 0.5556 nM 71 76 73 831.6667 nM 79 84 84 89 5.0000 nM 89 90 91 92 15.000 nM 92 91 90 93 IC₅₀(nM) 0.24 0.13 0.15 0.04

TABLE 21 Dose-dependent antisense inhibition of ‘Target 2’ mRNA in HUVECcells ISIS A ISIS B ISIS C ISIS D ISIS E 0.6173 nM 13 25 4 16 0 1.8519nM 16 12 19 22 21 5.5556 nM 39 37 41 41 39 16.6667 nM  60 61 62 70 6050.000 nM 71 75 68 78 71 150.000 nM  76 71 71 79 72 IC50 (nM) 13.3 12.514.9 9.0 17.3

TABLE 22 mRNA levels (% PBS control) of ‘Target 1’ and ‘Target 2’ atvarious time-points after treatment with actinomycin D or DRB Time‘Target 1’ ‘Target 2’ (hrs) actinomycin DRB actinomycin DRB 0.00 100 100100 100 0.25 92 82 106 131 0.5 102 90 101 106 0.75 88 98 99 108 1.00 82105 86 72 1.25 102 91 65 86 1.5 98 92 65 59 1.75 90 119 85 61 2.75 98 9860 37 3.75 101 98 43 41 4.75 99 108 39 19 5.75 92 113 23 9 6.75 85 10819 9 7.75 85 82 20 11 8.75 98 100 10 6

TABLE 23 mRNA levels of ‘Target 1’ and ‘Target 2’ at various time-pointsafter treatment with DRB or amanitin ‘Target 1’ ‘Target 2’ Time α- α-(hrs) DRB amanitin DRB amanitin 0.00 100 100 100 100 0.50 100 112 77 1090.75 100 113 59 107 1.00 107 118 55 117 1.25 110 118 33 111 1.50 99 11337 118 2.00 105 121 31 118 3.00 113 116 19 61 4.00 103 117 11 34 5.00106 113 8 25 6.00 101 114 8 13 7.00 93 115 8 14 8.00 92 109 11 12 9.0094 107 13 10Dose Response Studies with STAT3 Antisense Oligonucleotides andCorrelation RNA Stability in HUVEC Cells

The correlation of RNA stability of the non-nuclear-retained targetmolecule, STAT3, to the dose of antisense oligonucleotide required toachieve target knockdown was studied.

Antisense oligonucleotides, ISIS 337332 (GAAGCCCTTGCCAGCCATGT,designated herein as SEQ ID NO: 129), ISIS 345785 (TGCCTCCTCCTTGGGAATGT,designated herein as SEQ ID NO: 130), ISIS 455269 (GCTTAGATTCTCCTTAAACC,designated herein as SEQ ID NO: 131), ISIS 455271 (AAATGCTTAGATTCTCCTTA,designated herein as SEQ ID NO: 132), ISIS 455272 (TAAAATGCTTAGATTCTCCT,designated herein as SEQ ID NO: 133), ISIS 455291 (CAGCAGATCAAGTCCAGGGA,designated herein as SEQ ID NO: 134), ISIS 455370 (TAGGTGTTCCCATACGCACA,designated herein as SEQ ID NO: 135), ISIS 455371 (GCTAGGTGTTCCCATACGCA,designated herein as SEQ ID NO: 136), ISIS 455391 (TCAACTGTCTCCAGGCAGGA,designated herein as SEQ ID NO: 137), ISIS 455393 (CACATCAACTGTCTCCAGGC,designated herein as SEQ ID NO: 138), ISIS 455394 (GACACATCAACTGTCTCCAG,designated herein as SEQ ID NO: 139), ISIS 455411 (AACCCAATGGTAAGCCCAAG,designated herein as SEQ ID NO: 140), ISIS 455412 (TAAACCCAATGGTAAGCCCA,designated herein as SEQ ID NO: 141), ISIS 455471 (TGGAATTTGAATGCAGTGGC,designated herein as SEQ ID NO: 142), ISIS 455525 (GTACACACTATACACATTTT,designated herein as SEQ ID NO: 143), ISIS 455527 (GCCAAAAATTTACAACCCAT,designated herein as SEQ ID NO: 144), ISIS 455530 (AGAGACTAAAATCAAGGCTC,designated herein as SEQ ID NO: 145), ISIS 455536 (AGAACTGAAATTCCTTGGTC,designated herein as SEQ ID NO: 146), and 455540 (AAGTACTCTTTCAGTGGTTT,designated herein as SEQ ID NO: 147), all targeting human STAT3 RNAtranscript (the complement of GENBANK Accession NT_(—)010755.14truncated from nucleobases 4185000 to 4264000, designated herein as SEQID NO: 148) were tested at various doses. HUVEC cells were plated at adensity of 5,000 cells per well and transfected using Lipofectamine2000®reagent with 1.111 nM, 3.333 nM, 10 nM, and 30 nM concentrations of eachantisense oligonucleotide. After approximately 16 hours, RNA wasisolated from the cells and STAT3 RNA transcript levels were measured byquantitative real-time PCR. STAT3 RNA transcript levels were normalizedto total RNA content, as measured by RIBOGREEN®. Results are presentedin Table 24 as percent inhibition of STAT3, relative to untreatedcontrol cells.

All the gapmers of Table 24 are 5-10-5 gapmers, where the gap segmentcomprises ten 2′-deoxynucleosides and each wing segment comprises five2′-MOE nucleosides. The internucleoside linkages throughout each gapmerare phosphorothioate (P′S) linkages. All cytidine residues throughouteach gapmer are 5-methylcytidines.

To measure the stability of the STAT3 mRNA, HUVEC cells were treatedwith 8 μg/mL of actinomycin D over a period of 10 hours. Another set ofcells were treated with 75 μM5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) for the same timeperiod. Both RNA synthesis inhibitors gave similar results, as presentedin Table 25. Table 25 presents the percent mRNA levels compared to thePBS control after treatment with actinomycin D or DRB at different timepoints. The data demonstrates that STAT3 mRNA does not get degraded andis therefore stable for the period of treatment.

The data from Tables 24 and 25 demonstrates that the IC₅₀ foroligonucleotides targeting RNA transcripts which are stable is low,suggesting that these RNA transcripts are more amenable for targetingcompared to RNA transcripts which are not as stable. Therefore, theseantisense oligonucleotides will be useful to employ in cases whereoligonucleotide uptake by cells is low.

TABLE 24 Dose-dependent inhibition of STAT3 mRNA in HUVEC cells IC50ISIS No. 30.0 nM 10.0 nM 3.3333 nM 1.1111 nM (nM) 455540 87 65 28 8 6.5455536 85 62 21 5 7.6 455530 83 62 26 11 7.1 455527 87 60 21 0 7.8455525 72 42 9 0 13.4 455471 78 67 42 11 5.9 455371 90 73 46 15 4.5455370 78 53 23 6 9.0 455393 81 62 33 6 7.0 455391 75 54 30 10 8.5455394 85 63 33 5 6.7 455291 85 75 45 21 4.1 455271 86 71 40 16 4.9455272 86 57 30 28 5.7 455269 84 72 49 23 4.0 455411 82 58 21 10 7.9455412 79 50 27 15 8.4 345785 75 43 17 10 11.4 337332 80 49 12 0 10.5

TABLE 25 RNA levels (% PBS control) of STAT3 mRNA at various time-pointsafter treatment with actinomycin D or DRB Time (hrs) Actinomycin DRB0.25 97 111 0.50 105 104 0.75 101 69 1.00 93 103 1.75 97 97 2.00 104 963.00 95 104 4.00 104 90 5.00 102 81 6.00 97 94 7.00 98 84 8.00 92 919.00 101 84 10.00 98 85

Example 11 Antisense Inhibition of Murine MALAT1 in BALB/c Mice

ISIS 395251 (CCAGGCTGGTTATGACTCAG; target site 3338), incorporatedherein as SEQ ID NO: 96; ISIS 399462 (GGGTCAGCTGCCAATGCTAG, target site1280), incorporated herein as SEQ ID NO: 117; ISIS 399479(CGGTGCAAGGCTTAGGAATT, target site 4004), incorporated herein as SEQ IDNO: 118; and ISIS 399484 (ACAAGTACATTGGAGCACAT, target site 4206),incorporated herein as SEQ ID NO: 124; all targeting murine MALAT1 RNAtranscript (GENBANK Accession No. 3144_(—)097A, designated herein as SEQID NO: 125) and which demonstrated statistically significantdose-dependent inhibition in vitro, were evaluated for their ability toreduce murine MALAT1 RNA transcript in vivo.

Treatment

Male BALB/c mice were maintained on a 12-hour light/dark cycle and fedad libitum normal Purina mouse chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. Antisense oligonucleotides (ASOs) were prepared in bufferedsaline (PBS) and sterilized by filtering through a 0.2 micron filter.Oligonucleotides were dissolved in 0.9% PBS for injection.

The mice were divided into six treatment groups. The first four groupsreceived intraperitoneal injections of ISIS 395251, ISIS 399462, ISIS399479, or ISIS 399484 at a dose of 50 mg/kg twice per week for 3 weeks.The fifth group received intraperitoneal injections of controloligonucleotide ISIS 141923 at a dose of 50 mg/kg twice weekly for 3weeks. The sixth group received intraperitoneal injections of salinetwice weekly for 3 weeks. The saline-injected group served as thecontrol group to which the oligonucleotide-treated group was compared.

Inhibition of MALAT1 RNA

Twenty four hours after the final dose, the animals were sacrificed andliver tissue was isolated. Liver RNA was isolated for real-time PCRanalysis of MALAT1. As presented in Table 26, treatment with antisenseoligonucleotides reduced MALAT1 RNA transcript expression. The resultsare expressed as percent inhibition of MALAT1 RNA transcript, relativeto the PBS control. The control oligonucleotide, ISIS 141923, did notdemonstrate significant inhibition of MALAT1 RNA, as expected.

TABLE 26 Percent inhibition of MALAT1 RNA transcript in BALB/c mice %inhibition ISIS 141923 20 ISIS 395251 98 ISIS 399484 97 ISIS 399462 94ISIS 399479 96

Example 12 Dose-Dependent Antisense Inhibition of Murine MALAT1 RNA

ISIS 399462 and ISIS 399479, which showed statistically significant invivo inhibition of MALAT1, were further evaluated in a dose responsestudy.

Treatment

BALB/c mice were injected with 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS399462 or ISIS 399479 twice a week for 3 weeks. ISIS 141923 was injectedin another group of mice at 50 mg/kg twice a week for 3 weeks. A controlgroup of mice was injected with PBS twice a week for 3 weeks.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis ofMALAT1. As shown in Table 27, the antisense oligonucleotides achieveddose-dependent reduction of murine MALAT1 over the PBS control. Resultsare presented as percent inhibition of MALAT1, relative to the PBScontrol. The control oligonucleotide, ISIS 141923, did not demonstratesignificant inhibition of MALAT1 RNA, as expected.

TABLE 27 Dose-dependent inhibition of murine MALAT1 RNA transcript inBALB/c mice % mg/kg inhibition ISIS 141923 50 34 ISIS 399462 10 89 20 9540 97 ISIS 399479 10 96 20 94 40 96

Example 13 Dose-Dependent Antisense Inhibition of MALAT1 RNA in VariousMurine Tissues

ISIS 399462, which showed statistically significant in vivo inhibitionof MALAT1, was further evaluated in a dose response study.

Treatment

BALB/c mice were injected with 12.5 mg/kg, 25 mg/kg, or 50 mg/kg of ISIS399462 twice a week for 3.5 weeks. A control group of mice was injectedwith PBS twice a week for 3.5 weeks.

RNA Analysis

RNA was extracted from liver, heart, tibialis anterior (TA), diaphragm,quadriceps, and gastrocnemius muscle tissues for real-time PCR analysisof MALAT1. As shown in Table 28, ISIS 399462 achieved dose-dependentreduction of murine MALAT1 in all tissues over the PBS control. Resultsare presented as percent inhibition of MALAT1, relative to the PBScontrol.

TABLE 28 Dose-dependent inhibition of murine MALAT1 RNA transcript invarious murine tissues 12.5 mg/kg 25 mg/kg 50 mg/kg Liver 94 95 96 Heart31 50 54 TA 61 74 80 Diaphragm 63 73 83 Quadriceps 64 84 85Gastrocnemius 70 79 89

Example 17 Dose Response Studies of Antisense Oligonucleotides in MHT2WTumor Cells

Potency of antisense oligonucleotides in MHT2W cells was studied.

MHT2W cells were plated at a density of 4,500 cells per well in a96-well plate. The cells were treated the next day with 0.02 μM, 0.1 μM,0.5 μM, 2.5 μM, and 10 μM concentrations of ISIS 15 (5-10-5 MOE gapmertargeting SR-B1), ISIS 19 (5-10-5 MOE gapmer targeting MALAT-1), ISIS 20(5-10-5 MOE gapmer targeting MALAT-1), ISIS 21 (3-10-3 (S)-cEt gapmertargeting STAT3), or ISIS 22 (5-10-5 MOE gapmer targeting STAT3). Afterapproximately 16 hours, RNA was isolated from the cells and target mRNAtranscript levels were measured by quantitative real-time PCR. Eachtarget mRNA transcript levels were normalized to total RNA content, asmeasured by RIBOGREEN®. Results are presented in Table 29 as percentinhibition of SR-B1 or PTEN, relative to untreated control cells.

The 5-10-5 gapmers in Table 29 are oligonucleotides where the gapsegment comprises ten 2′-deoxynucleosides and each wing segmentcomprises five 2′-MOE nucleosides. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P═S) linkages. All cytidineresidues throughout each gapmer are 5-methylcytidines. The 3-10-3gapmers are oligonucleotides where the gap segment comprises ten2′-deoxynucleosides and each wing segment comprises threedeoxynucleosides linked to (S)-cEt sugars.

TABLE 29 Dose-dependent antisense inhibition of target mRNA in MHT2Wtumor cells ISIS 0.02 0.1 0.5 2.5 10 IC₅₀ No Target Motif μM μM μM μM μM(μM) 15 SR-B1 5-10-5 37 53 61 72 82 1.00 MOE 19 MALAT-1 5-10-5 16 47 6362 72 0.40 MOE 20 MALAT-1 5-10-5 44 64 73 79 85 0.02 MOE 21 STAT3 3-10-39 8 25 42 61 5.24 (S)-cEt 22 STAT3 5-10-5 4 16 17 13 24 >10 MOE

Example 18 Dose Response Studies of Antisense Oligonucleotides in HumanGlioblastoma Tumor Cells

Potency of antisense oligonucleotides in the human glioblastoma celllines, SNB19 and U251, was studied.

SNB19 cells were plated at a density of 4,500 cells per well in a96-well plate. The cells were treated the next day with 0.02 μM, 0.1 μM,0.5 μM, 2.5 μM and 10 μM concentrations of ISIS 23 (5-10-5 MOE gapmertargeting STAT3), ISIS 21 (3-10-3 (S)-cEt gapmer targeting STAT3), ISIS24 (5-10-5 MOE gapmer targeting STAT3), ISIS 20 (5-10-5 MOE gapmertargeting MALAT-1), or ISIS 25 (5-10-5 MOE gapmer targeting kinesinfamily member 11 or Eg5). After approximately 16 hours, RNA was isolatedfrom the cells and target mRNA transcript levels were measured byquantitative real-time PCR. Each target mRNA transcript levels werenormalized to total RNA content, as measured by RIBOGREEN®. Results arepresented in Table 30 as percent inhibition of STAT3, MALAT-1 or Eg5,relative to untreated control cells.

U251 cells were plated at a density of 4,500 cells per well in a 96-wellplate. The cells were treated the next day with 0.02 μM, 0.1 μM, 0.5 μM,2.5 μM and 10 μM concentrations of ISIS 23 (5-10-5 MOE gapmer targetingSTAT3), ISIS 21 (3-10-3 (S)-cEt gapmer targeting STAT3), ISIS 24 (5-10-5MOE gapmer targeting STAT3), ISIS 20 (5-10-5 MOE gapmer targetingMALAT-1), or ISIS 25 (5-10-5 MOE gapmer targeting kinesin family member11 or Eg5). After approximately 16 hours, RNA was isolated from thecells and target mRNA transcript levels were measured by quantitativereal-time PCR. Each target mRNA transcript levels were normalized tototal RNA content, as measured by RIBOGREEN®. Results are presented inTable 31 as percent inhibition of STAT3, MALAT-1 or Eg5, relative tountreated control cells.

The 5-10-5 gapmers in Tables 30 and 31 are oligonucleotides where thegap segment comprises ten 2′-deoxynucleosides and each wing segmentcomprises five 2′-MOE nucleosides. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P═S) linkages. All cytidineresidues throughout each gapmer are 5-methylcytidines. The 3-10-3gapmers are oligonucleotides where the gap segment comprises ten2′-deoxynucleosides and each wing segment comprises threedeoxynucleosides linked to (S)-cEt sugars.

TABLE 30 Dose-dependent antisense inhibition of target mRNA in SNB19glioblastoma cells ISIS 0.02 0.1 0.5 2.5 10 IC₅₀ No Target Motif μM μMμM μM μM (μM) 20 MALAT-1 5-10-5 43 82 94 95 97 0.008 MOE 21 STAT3 3-10-31 34 55 81 92 0.4 (S)-cEt 23 STAT3 5-10-5 2 9 46 69 72 1.1 MOE 24 STAT35-10-5 24 43 51 79 91 0.2 MOE 25 Eg5 5-10-5 30 38 46 61 71 0.5 MOE

TABLE 31 Dose-dependent antisense inhibition of target mRNA in U251glioblastoma cells ISIS 0.02 0.1 0.5 2.5 10 IC₅₀ No Target Motif μM μMμM μM μM (μM) 20 MALAT-1 5-10-5 31 51 53 64 88 0.19 MOE 21 STAT3 3-10-31 1 4 27 52 2.8 (S)-cEt 23 STAT3 5-10-5 4 6 4 7 24 >10 MOE 24 STAT35-10-5 0 0 0 28 31 >10 MOE 25 Eg5 5-10-5 0 1 11 14 19 >10 MOE

Example 19 In Vivo Studies with Antisense Oligonucleotides in MHT2WXenograft Tumor Model

In vivo uptake and potency of an antisense oligonucleotide targeting anuclear-retained RNA was compared with that of antisense oligonucleotidetargeting non-nuclear retained RNAs in a xenograft tumor model.

Treatment

MHT2W tumor cells (1×10⁶, of human origin) were injected subcutaneouslyin female Balb/c nu/nu mice. After 4-7 days, ISIS 395251(CCAGGCTGGTTATGACTCAG (SEQ ID NO: 96), targeting nuclear-retained RNA,MALAT1) or ISIS 383741 (GACTCTTGCAGGAATCGGCT (SEQ ID NO: 149), targetingnon-nuclear-retained RNA, Stat3) at a dose of 50 mg/kg was injectedintraperitoneally two times per week for a total of 7 doses. The micewere euthanized one day after the last dose.

mRNA Analysis

RNA was isolated from the liver and tumor cells at the end of the studyfor real-time PCR analysis of MALAT1 and Stat3, and normalized toRIBOGREEN®. The results are presented in Table 32 expressed as percentinhibition of mRNA transcript, relative to the PBS control.

TABLE 32 Percent inhibition of mRNA transcript after antisense treatmentin a xenograft tumor mouse model ISIS No. Tumor Liver 395251 49 93383741 27 93

Example 23 In Vivo Studies of Antisense Oligonucleotide Pharmacology inMice Bone Marrow Cells

In vivo uptake and potency of antisense oligonucleotides was studied inbone marrow cells of C57Bl/6 mice.

Treatment

Groups of mice were treated with ISIS 19 (5-10-5 MOE gapmer targetingMALAT-1), ISIS 23 (5-10-5 MOE gapmer targeting STAT3) or ISIS 15 (5-10-5MOE gapmer targeting SR-B1) at a dose of 40 mg/kg injected intravenouslydaily for 4 days. A control group of mice was injected with PBSintravenously daily for 4 days. The mice were euthanized one day afterthe last dose. Bone marrow and liver tissue was collected from the mice.CD45⁺ leukocytes were isolated from the bone marrow for analysis.

mRNA Analysis

RNA was isolated from the liver, bone marrow cells and bone marrow CD45⁺leukocytes at the end of the study for real-time PCR analysis ofMALAT-1, STAT3 and SR-B1, and normalized to RIBOGREEN®. Table 33presents the percent inhibition of the murine RNA transcript, relativeto the PBS control. The data demonstrates that antisenseoligonucleotides targeting SR-B1 and STAT3, although potent in theliver, are not able to diffuse into tumor cells easily. Antisenseoligonucleotides targeting a nuclear-retained target, MALAT-1, were ableto diffuse into the bone marrow cells and cause potent inhibition oftarget RNA.

TABLE 33 Percent inhibition of RNA transcript after antisense treatmentISIS Bone CD45⁺ No Target Motif Liver marrow cells 15 SR-B1 5-10-5 93 160 MOE 19 MALAT-1 5-10-5 95 53 49 MOE 23 STAT3 5-10-5 80 0 0 MOE

Example 24 Antisense Inhibition of Human Alpha1 Skeletal Actin in HepG2Cells

Antisense oligonucleotides targeted to a human alpha1 skeletal actinnucleic acid were tested for their effect on alpha1 actin RNA transcriptin vitro. Cultured HepG2 cells at a density of 20,000 cells per wellwere transfected using electroporation with 10,000 nM antisenseoligonucleotide. After approximately 24 hours, RNA was isolated from thecells and alpha1 actin RNA transcript levels were measured byquantitative real-time PCR. Alpha1 actin RNA transcript levels wereadjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of alpha1 actin, relative tountreated control cells.

The antisense oligonucleotides in Table 34 are 5-10-5 gapmers, where thegap segment comprises ten 2′-deoxynucleosides and each wing segmentcomprises five 2′-MOE nucleosides. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P═S) linkages. All cytidineresidues throughout each gapmer are 5-methylcytidines. ‘Target startsite’ indicates the 5′-most nucleotide to which the antisenseoligonucleotide is targeted. ‘Target stop site’ indicates the 3′-mostnucleotide to which the antisense oligonucleotide is targeted. All theantisense oligonucleotides listed in Table 34 target SEQ ID NO: XXX(GENBANK Accession No. NM_(—)001100.3).

TABLE 34 Inhibition of human alpha1 actin RNA transcriptin HepG2 cells by 5-10-5 gapmers targeting SEQ ID NO: 177 Target Target% SEQ Start Stop ISIS inhibi- ID Site Site No Sequence tion NO. 16 35445205 AGCGAGGCTTCACTTGGCGC 74 150 20 39 190403 GGGAAGCGAGGCTTCACTTG 75151 1028 1047 190401 GCGGTCAGCGATCCCAGGGT 78 152 1058 1077 445225GGGTGCCAGCGCGGTGATCT 73 153 1320 1339 445231 TGTTACAAAGAAAGTGACTG 74 1541339 1358 445232 CGATGGCAGCAACGGAAGTT 96 155 1348 1367 445233GTCAGTTTACGATGGCAGCA 100 156 1417 1436 445235 CAGGGCTTTGTTTCGAAAAA 91157 1430 1449 445236 CCATTTTCTTCCACAGGGCT 99 158 1447 1466 445237ATGCTTCTTCAAGTTTTCCA 97 159 1460 1479 445238 CAGAATGACTTTAATGCTTC 95 160

Example 25 Dose-Dependent Antisense Inhibition of Human Alpha1 Actin inHepG2 Cells

Several of the antisense oligonucleotides exhibiting in vitro inhibitionof alpha1 actin in HepG2 cells (see Example 26) were tested at variousdoses. Cells were plated at a density of 20,000 cells per well andtransfected using electroporation with 625 nM, 1,250 nM, 2,500 nM, 5,000nM, 10,000 nM and 20,000 nM concentrations of each antisenseoligonucleotide. After approximately 16 hours, RNA was isolated from thecells and alpha1 actin RNA transcript levels were measured byquantitative real-time PCR using primer probe set RTS3154 (forwardsequence CCACCGCAAATGCTTCTAGAC, designated herein as SEQ ID NO: 161;reverse sequence CCCCCCCATTGAGAAGATTC, designated herein as SEQ ID NO:162; probe sequence CTCCACCTCCAGCACGCGACTTCTX, designated herein as SEQID NO: 163). Alpha1 actin RNA transcript levels were normalized to totalRNA content, as measured by RIBOGREEN®. Results are presented in Table35 as percent inhibition of alpha1 actin, relative to untreated controlcells.

TABLE 35 Dose-dependent antisense inhibition of human alpha1 actin inHepG2 cells ISIS 625.0 1250.0 2500.0 5000.0 10000.0 20000.0 IC₅₀ No. nMnM nM nM nM nM (mM) 445233 21 72 63 82 96 83 1.1 445236 26 68 82 91 9091 0.8 445237 36 59 76 84 83 90 0.8 445232 14 42 54 59 80 91 2.6 44523827 43 54 73 76 90 2 445235 26 52 29 58 59 24 n.a. 190403 25 29 36 25 6154 n.a. 190401 17 14 40 68 76 72 3.9 445225 25 23 49 28 52 50 n.a.445205 26 31 34 28 55 36 n.a. 445231 30 25 39 26 42 36 n.a

Example 26 In Vivo Antisense Inhibition of Human Alpha1 Actin inTransgenic Mice

HSA (human skeletal actin)^(LR) (long repeat) mice were generated byinsertion in FVB/N mice of a transgene with 250 CTG repeats in the 3′UTR of human skeletal actin. The transgene is expressed in the mice as aCUG repeat RNA, which is retained in the nucleus, forming nuclearinclusions or foci, similar to that seen in human tissue samples ofpatients with myotonic dystrophy.

ISIS 190403, ISIS 445236 and ISIS 445238, which demonstratedstatistically significant dose-dependent inhibition in vitro, wereevaluated for their ability to reduce human alpha1 actin RNA transcriptin vivo.

Treatment

HSA^(LR) mice were maintained on a 12-hour light/dark cycle and fed adlibitum normal Purina mouse chow. Animals were acclimated for at least 7days in the research facility before initiation of the experiment.Antisense oligonucleotides (ASOs) were prepared in PBS and sterilized byfiltering through a 0.2 micron filter. Oligonucleotides were dissolvedin 0.9% PBS for injection.

The mice were divided into four treatment groups. The first three groupsreceived subcutaneous injections of ISIS 190403, ISIS 445236 or ISIS445238 at a dose of 25 mg/kg twice per week for 4 weeks. The fourthgroup received subcutaneous injections of PBS twice weekly for 4 weeks.The PBS-injected group served as the control group to which theoligonucleotide-treated group was compared.

Inhibition of Alpha1 Actin RNA

Twenty four hours after the final dose, the animals were sacrificed andtissue from the quadriceps muscles (left and right), gastrocnemiusmuscles (left and right), and tibialis anterior muscles (left and right)was isolated. RNA was isolated for real-time PCR analysis of alpha1actin and normalized to 18s RNA. As presented in Table 36, treatmentwith antisense oligonucleotides reduced human alpha1 actin RNAtranscript expression. The results are expressed as percent inhibitionof alpha1 actin transcript, relative to the control.

TABLE 36 Percent inhibition of human alpha1 actin RNA transcript inHSA^(LR) mice ISIS ISIS ISIS Muscle Type 190403 445236 445238 Quadriceps16 83 72 Gastrocnemius 0 85 73 Tibialis anterior 2 81 71

Fluorescence in Situ Hybridization of Alpha1 Actin in Muscles

Frozen muscle tissue sections were fixed in fresh 3% paraformaldehyde inPBS solution for 15-20 minutes, after which they were rinsed twice withPBS for 5 minutes. The nuclei were permeabilized with 0.5% Triton X-100for 5 minutes after which the tissue was blocked with normal goat serumfor 30 minutes. The sections were incubated a 2′-O-methyl RNA targetedto alpha1 actin that is 5′-labeled with Texas Red (Integrated DNATechnologies). The sections were counter-stained with DAPI to label thenuclei. The sections were mounted and viewed with a standardfluorescence microscope. Image acquisition was by Metavue software anddeconvolution was achieved by Autoquant software.

All muscle tissue sections from mice treated with ISIS 445236 and ISIS445238 displayed reduced fluorescent intensity of alpha1 actin signal atthe ribonuclear foci, indicating antisense inhibition of human alpha1actin mRNA and reduction of the RNA in the nuclear foci.

Assessment of Myotonia by Electromyography

Myotonia refers to repetitive action potential that is due to delayedrelaxation of muscle fibers. This phenomenon is observed in patients ofmyotonic dystrophy as well as in the HSA^(LR) mice. When the EMG needleis inserted into a myotonic muscle, the electrical activity is prolongedfor up to several seconds past when the insertional activity shouldnormally cease. The frequency of myotonic discharges ranges from 50 to100 impulses per second.

Myotonia may be measured via electromyography and is graded in thefollowing manner: grade 0 refers to no myotonia elicited by any needleinsertion (0%); grade 1 refers to myotonia elicited by less than 50%needle insertions; grade 2 refers to myotonia elicited by more than 50%needle insertions; and grade 3 refers to mytonia elicited by 100% needleinsertions.

Before electromyography, mice were anesthetized by using i.p. 100 mg_kgketamine, 10 mg_kg xylazine, and 3 mg_kg acepromazine or 250 mg_kg 2,2,2tribromoethanol. Electromyography on left and right quadriceps, left andright gastrocnemius muscles, left and right tibialis anterior musclesand lumbar paraspinals muscles was performed as previously described(Kanadia et al, 2003, Science, 302: 1978-1980) by using 30 gaugeconcentric needle electrodes and a minimum of 10 needle insertions foreach muscle. The data is presented in Table 37 as the average myotoniagrade observed in four mice of each group and demonstrates significantreduction of myotonia in mice treated with ISIS 445236 and ISIS 445238.

TABLE 37 Average reduction of myotonia in various muscles of antisenseoligonucleotide-treated HSA^(LR) mice ISIS ISIS ISIS PBS 190403 445236445238 Left quadriceps 3.00 3.00 0.00 0.25 Right quadriceps 3.00 3.000.00 0.00 Left gastrocnemius 3.00 3.00 0.00 0.25 Right gastrocnemius3.00 3.00 0.00 0.25 Left Tibialis anterior 2.75 2.50 0.00 0.00 RightTibialis anterior 2.75 2.50 0.00 0.00 Lumbar paraspinals 3.00 3.00 0.000.75

Example27 Dose-Dependent Inhibition of Long CUG Repeat mRNA (HSA^(LR)Mice) and a Short CUG Repeat (HSA^(SR) Mice) by SubcutaneousAdministration in Transgenic Mice

Dose-dependent inhibition of mRNA transcripts containing a long CUGrepeat (HSA^(LR) mice) and a short CUG repeat (HSA^(SR) mice), wasevaluated. HSA-short repeat (HSA^(SR)) mice express the identicaltransgene as the HSA^(LR) mice, except that 5 instead of 250 CUG repeatsare inserted in the 3′ UTR. HSA^(SR) mice do not have myotonia, splicingchanges, or any other observable myotonia phenotype. ISIS 445236 wasused in this assay.

Treatment

HSA^(LR) mice were divided into four treatment groups. The first threegroups received subcutaneous injections of ISIS 445236 at doses of 2.5mg/kg, 8.5 mg/kg or 25 mg/kg twice per week for 4 weeks. The fourthgroup received subcutaneous injections of PBS twice per week for 4weeks. The PBS-injected group served as the control group to which theoligonucleotide-treated group was compared. HSA^(SR) mice were alsodivided into four groups and similarly treated.

Inhibition of Alpha1 Actin RNA

Twenty four hours after the final dose, the animals were sacrificed andtissue from the quadriceps muscles (left and right), gastrocnemiusmuscles (left and right), and tibialis anterior muscles (left and right)was isolated. RNA was isolated for real-time PCR analysis of alpha1actin and normalized to 18s RNA. The results are presented in Tables 38and 39 and are expressed as percent inhibition of alpha1 actintranscript, relative to the control. Greater inhibition of thenuclear-retained long repeat in the muscle of HSA^(LR) mice was achievedcompared with the non-nuclear-retained short repeat in the muscle ofHSA^(SR) mice.

TABLE 38 Percent inhibition of human alpha1 actin RNA transcript inHSA^(LR) mice Dose Tibialis (mg/kg) Quadriceps Gastrocnemius anterior2.5 24 36 46 8.5 53 66 59 25 86 86 90

TABLE 39 Percent inhibition of human alpha1 actin RNA transcript inHSA^(SR) mice Dose Tibialis (mg/kg) Quadriceps Gastrocnemius anterior2.5 15 14 0 8.5 30 11 0 25 59 48 54

Example 28 Dose Response Studies with Antisense OligonucleotidesTargeting Human Dystrophia Myotonica-Protein Kinase (DMPK) in DM1Fibroblast Cells

The mutant form of the DMPK mRNA, harboring large CUG repeats, are fullytranscribed and polyadenylated, but remained trapped in the nucleus(Davis et al, 1997, Proc. Natl. Acad. Sci. U.S.A. 94, 7388-7393). Thesemutant nuclear-retained mRNA are one of the most important pathologicalfeatures of myotonic dystrophy 1 (DM1). Antisense inhibition of mutantDMPK mRNA in DM1 fibroblast cells was studied.

DM1 fibroblast cells were plated at a density of 4,500 cells per welland transfected using Cytofectin reagent with 9.375 nM, 18.75 nM, 37.5nM, 75 nM, 150 nM, and 300 nM concentrations of each antisenseoligonucleotide. After approximately 16 hours, RNA was isolated from thecells and DMPK RNA transcript levels were measured by quantitativereal-time PCR using primer probe set RTS3164 (forward sequenceAGCCTGAGCCGGGAGATG, designated herein as SEQ ID NO: 164; reversesequence GCGTAGTTGACTGGCGAAGTT, designated herein as SEQ ID NO: 165; andprobe sequence AGGCCATCCGCACGGACAACCX, designated herein as SEQ ID NO:166). DMPK RNA transcript levels were normalized to total RNA content,as measured by RIBOGREEN®. Results are presented in Table 41 as percentinhibition of DMPK, relative to untreated control cells.

An assay with similar conditions was also performed with primer probeset RTS3162 (forward sequence CGGGCCGTCCGTGTT, designated herein as SEQID NO: 167; reverse sequence CTTTGCACTTTGCGAACCAA, designated herein asSEQ ID NO: 168; and probe sequence CATCCTCCACGCACCCCCACCX, designatedherein as SEQ ID NO: 169), which targets the 3′-end of the DMPKtranscript. Results are presented in Table 42 as percent inhibition ofDMPK, relative to untreated control cells.

All the gapmers assayed are described in Table 40 and are 5-10-5gapmers, where the gap segment comprises ten 2′-deoxynucleosides andeach wing segment comprises five 2′-MOE nucleosides. The internucleosidelinkages throughout each gapmer are phosphorothioate (P═S) linkages. Allcytidine residues throughout each gapmer are 5-methylcytidines. All thegapmers target SEQ ID NO: 170 (the complement of GENBANK Accession No.NT_(—)011109.15 truncated at nucleotides 18540696 to 18555106). ‘Targetstart site’ indicates the 5′-most nucleotide to which the antisenseoligonucleotide is targeted. ‘Target stop site’ indicates the 3′-mostnucleotide to which the antisense oligonucleotide is targeted.

TABLE 40 Chimeric antisense oligonucleotides targetinghuman dystrophia myotonica-protein kinase (SEQ ID NO: 198) Target TargetSEQ Start Stop ISIS ID Site Site No. Sequence NO. 812 831 299471TGCTCCCGACAAGCTCCAGA 171 13553 13572 444401 TTGCACTTTGCGAACCAACG 17213562 13581 444404 AAGAAAGCTTTGCACTTTGC 173 13748 13767 444436GTCGGAGGACGAGGTCAATA 174 13226 13245 445569 CGGAGCGGTTGTGAACTGGC 175

TABLE 41 Dose-dependent antisense inhibition of DMPK mRNA in DM1fibroblast cells with RTS3164 ISIS 9.375 18.75 37.5 75.0 150.0 300.0IC₅₀ No. nM nM nM nM nM nM nM 299471 10 25 31 47 61 73 86.3 444401 8 2741 60 67 74 64.3 444404 10 21 31 43 55 73 100 444436 7 17 36 64 68 7072.3 445569 19 31 41 59 46 77 72.2

TABLE 42 Dose-dependent antisense inhibition of DMPK mRNA in DM1fibroblast cells with RTS3162 ISIS 9.375 18.75 37.5 75.0 150.0 300.0IC₅₀ No nM nM nM nM nM nM (nM) 299471 7 25 29 46 48 69 115.3 444401 2034 52 72 83 89 35.8 444404 5 20 28 42 54 77 98.8 444436 12 15 27 61 6875 74.3 445569 5 25 33 53 50 76 89.6

Example 29 In Vivo Antisense Inhibition of Human DMPK in Transgenic Mice

LC15 mice, Line A, are transgenic mice containing the entire human DMPK3′UTR (developed by Wheeler et al, University of Rochester). The miceare the second generation of mice backcrossed to an FVB background. Thetransgene is expressed in the mice as a CUG repeat RNA, which isretained in the nucleus, forming nuclear inclusions or foci, similar tothat seen in human tissue samples of patients with myotonic dystrophy(DM1). There are 350-400 CUG repeats in the DMPK transgene. These micedisplay early signs of DM1 and do not display any myotonia in theirmuscle tissues.

ISIS 445569, ISIS 444404, ISIS 444436 and ISIS 473810, whichdemonstrated statistically significant dose-dependent inhibition invitro (see Example 5), were evaluated for their ability to reduce humanDMPK RNA transcript in vivo.

Treatment

LC15, Line A mice were maintained on a 12-hour light/dark cycle and fedad libitum normal Purina mouse chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. Antisense oligonucleotides (ASOs) were prepared in PBS andsterilized by filtering through a 0.2 micron filter. Oligonucleotideswere dissolved in 0.9% PBS for injection.

The mice were divided into five treatment groups. The first three groupsreceived subcutaneous injections of ISIS 445569, ISIS 444404 or ISIS444436 at a dose of 25 mg/kg twice per week for 4 weeks. The fourthgroup received subcutaneous injections of ISIS 473810 at a dose of 12.5mg/kg twice per week for 4 weeks. The fifth group received subcutaneousinjections of PBS twice weekly for 4 weeks. The PBS-injected groupserved as the control group to which the oligonucleotide-treated groupwas compared.

Inhibition of DMPK RNA

Twenty four hours after the final dose, the animals were sacrificed andtissue from the quadriceps muscles was isolated. RNA was isolated forreal-time PCR analysis of DMPK and normalized to 18s RNA. As presentedin Table 43, treatment with antisense oligonucleotides reduced humanDMPK RNA transcript expression. The results are expressed as percentinhibition of DMPK transcript, relative to the PBS control.

TABLE 43 Antisense inhibition of human DMPK RNA transcript in LC15 mice% ISIS No mg/kg/wk inhibition 444404 50 20 444404 50 55 444436 50 41473810 25 56

Assessment of Myotonia by Electromyography

Electromyography on left and right quadriceps, left and rightgastrocnemius muscles, left and right tibialis anterior muscles andlumbar paraspinals muscles was performed as previously described(Kanadia et al, 2003, Science, 302: 1978-1980) by using 30 gaugeconcentric needle electrodes and a minimum of 10 needle insertions foreach muscle. Since LC15 mice do not have myotonia, neither the controlgroup nor the treatment groups displayed any myotonia in any muscletested.

Example 30 In Vivo Antisense Inhibition of Human DMPK in Transgenic Mice

LC15 mice, Line D, are transgenic mice containing the entire human DMPK3′UTR (developed by Wheeler et al, University of Rochester). The miceare the third generation of mice backcrossed to an FVB background. Thetransgene is expressed in the mice as a CUG repeat RNA, which isretained in the nucleus, forming nuclear inclusions or foci, similar tothat seen in human tissue samples of patients with myotonic dystrophy(DM1). There are 350-400 CUG repeats in the DMPK transgene. These micedisplay early signs of DM1 and do not display any myotonia in theirmuscle tissues.

ISIS 445569, ISIS 444404, ISIS 444436 and ISIS 473810 were furtherevaluated for their ability to reduce human DMPK RNA transcript in vivo.

Treatment

LC15, Line A mice were maintained on a 12-hour light/dark cycle and fedad libitum normal Purina mouse chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. Antisense oligonucleotides (ASOs) were prepared in PBS andsterilized by filtering through a 0.2 micron filter. Oligonucleotideswere dissolved in 0.9% PBS for injection.

The mice were divided into six treatment groups. The first three groupsreceived subcutaneous injections of ISIS 445569, ISIS 444404 or ISIS444436 at a dose of 25 mg/kg twice per week for 4 weeks. The fourthgroup received subcutaneous injections of ISIS 473810 at a dose of 12.5mg/kg twice per week for 4 weeks. The fifth group received subcutaneousinjections of ISIS 473810 at a dose of 6.25 mg/kg twice per week for 4weeks. The sixth group received subcutaneous injections of PBS twiceweekly for 4 weeks. The PBS-injected group served as the control groupto which the oligonucleotide-treated group was compared.

Inhibition of DMPK RNA

Twenty four hours after the final dose, the animals were sacrificed andtissue from the quadriceps muscles was isolated. RNA was isolated forreal-time PCR analysis of DMPK and normalized to 18s RNA. As presentedin Table 44, treatment with antisense oligonucleotides reduced humanDMPK RNA transcript expression. The results are expressed as percentinhibition of DMPK transcript, relative to the PBS control.

TABLE 44 Antisense inhibition of human DMPK RNA transcript in LC15 mice% ISIS No mg/kg/wk inhibition 444404 50 24 444404 50 30 444436 50 17473810 25 7 473810 12.5 18

Assessment of Myotonia by Electromyography

Electromyography on left and right quadriceps, left and rightgastrocnemius muscles, left and right tibialis anterior muscles andlumbar paraspinals muscles was performed as previously described(Kanadia et al, 2003, Science, 302: 1978-1980) by using 30 gaugeconcentric needle electrodes and a minimum of 10 needle insertions foreach muscle. Since LC15 mice do not have myotonia, neither the controlgroup nor the treatment groups displayed any myotonia in any muscletested.

Example 31 In Vivo Antisense Inhibition of Human DMPK in SXL TransgenicMouse Model

Using hDMPK-targeting ASOs 444401 and 299471 target knockdown in soleusmuscle was measured in SXL mice. The SXL mouse is transgenic for theentire DMPK gene and promoter and contains a 1000 CUG repeat sequence inthe 3′UTR of DMPK gene. Mice were dosed 50 mg/kg twice weekly for 4weeks (n=3 mice per group, except n=2 for saline-injected controls).Results of Taqman assays are shown in FIG. 1 for mut-hDMPK mRNA (FIG.1A) and endogenous mouse Dmpk mRNA (FIG. 1B).

Example 32 In Vivo Inhibition of snoRNAs

ISIS 462026 (targeting U16) and ISIS 477499 (targeting U50),demonstrating significant inhibition of their respective snoRNAs, weretested in mice and the efficacy of the gapmers was evaluated.

Treatment

Two groups of five seven-week old balb-c mice were each administeredsubcutaneously with 100 mg/kg of ISIS 462026 or ISIS 477499. Anothergroup of five mice was injected with 100 mg/kg of controloligonucleotide ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, designated herein asSEQ ID NO: 176). Another group of five mice were injected subcutaneouslywith PBS. The mice injected with PBS served as a control group. The micewere sacrificed 72 hours later and several tissues were harvested fortarget mRNA analysis. Tissues harvested were: liver, heart, spleen,white adipose tissue (WAT), kidney, and muscle.

RNA Analysis

Total RNA from each of the various tissues was separately prepared usingTri-Reagent, based on the manufacturer's instructions. Five microgramsof total RNA was separated in 8% polyacrylamide-7M urea gels and wastransferred onto a membrane, using semi-dry transfer apparatus. Northernhybridization was performed using U16 snoRNA-specific 5′-end labeledoligonucleotide probe (5′-TTGCTCAGTAAGAATTTTCG-3′, designated herein asSEQ ID NO: 177), and U50 snoRNA-specific 5′-end labeled oligonucleotideprobe (5′-GGTTCGGGATAAGATCATCACA-3′, designated herein as SEQ ID NO:178). U2 snRNA were detected and served as a control for loading. Thedensity of the bands was scanned using an ImageJ densitometer. Resultsfor inhibition are presented in FIG. 2. The data indicates that ISIS462026 and ISIS 477499 significantly inhibited their target snoRNAexpression.

Evaluation of rRNA Methylation

Total RNA from liver samples was pooled for each group and subjected toprimer extension analysis to detect rRNA methylation at positions A485in 18S rRNA, targeted by U16 snoRNA, or C2613 in 28S rRNA, targeted byU50 snoRNA. The results are presented in Table 45 and demonstratesignificant inhibition at 0.05 mM dNTP concentration, compared to thePBS control.

TABLE 45 Inhibition of rRNA methylation by antisense oligonucleotides inmouse livers relative to the PBS control % inhibition ISIS of rRNA Nomethylation 462026 >95 477499 >93

Example 33 In Vitro Inhibition of lincRNA_SFPQE, lincRNA_p21,lincRNA_HOXA1, HOTAIR, PCGEM1, and MIAT mRNA

Antisense oligonucleotides were designed targeting lincRNA_SFPQE,lincRNA_p21, lincRNA_HOXA1, HOTAIR, PCGEM1, and MIAT mRNA sequences. Theantisense oligonucleotides were tested in vitro. Various doses ofantisense oligonucleotides were individually tested in various cells andthe mRNA expression levels of the corresponding targets were analyzed byRT-PCR.

The results indicated that the antisense oligonucleotides fornuclear-retained RNAs, such as lincRNA_SFPQE, lincRNA_p21,lincRNA_HOXA1, HOTAIR, PCGEM1, and MIAT, were able to reduce theirtarget mRNA sequences with a potency similar to that demonstrated byantisense oligonucleotides targeting MALAT1 (see Examples 1-6).

1.-69. (canceled)
 70. A method of achieving a pharmacologically relevantreduction of a nuclear-retained RNA in a cell or tissue having lowantisense oligonucleotide uptake, comprising administering to an animalsuspected of having said nuclear-retained RNA a chemically-modifiedantisense oligonucleotide complementary to said nuclear-retained RNA inan amount effective to activate a nuclear ribonuclease capable ofcleaving the nuclear-retained RNA to achieve said pharmacologicallyrelevant reduction.
 71. The method of claim 70, wherein saidnuclear-retained RNA is associated with a disease or condition in saidtissue and wherein said animal is selected as having said disease orcondition.
 72. The method of claim 70, wherein said nuclear-retained RNAis a non-coding RNA.
 73. The method of claim 72, wherein said non-codingRNA is a long non-coding RNA, short non-coding RNA, large interveningnon-coding RNA, repeat element containing RNA, expanded nucleotiderepeat-containing RNA, snoRNA, scaRNA, or enrRNA.
 74. The method ofclaim 72 wherein said non-coding RNA is any of Xlsirt, Satellite III,Hox C5 transcript variant 2 (non-coding), Menβ, Neat1, Neat2, hsr-omega,hothead, Kit, Xist, Air, Tsix, Mirg, Kcnq1ot1, AK045070, P-rex1,ZNF127AS, NESPAS, SRG1, Hotair, Gomafu, Sox2ot, Rian, CAT2, Xite, Jpx,Ftx, RoX1, RoX2, H19, Igf2, IPW, UBE3A, ATP10C, pgc, 7SK, RNA Pol IItranscription elongation factor P-TEFb, B2, HSR-1, BC1, BC200, NRSE,NRON, NFAT transcription factor, Makorin-p1, HAR1F, HAR1R, OCC1,DD3/PCA3, PCGEM1, NCRMS, HIS-1, BCMS, CMPD, NC612, SRA, DISC2,PSZA11q14, RAY1/ST7, UBE3A-AS, SCA8, 22k48, C6orf37OS, COPG2IT1, DGCR5,KCNQ1 overlapping transcript 1 (non-protein coding), MESTIT 1, andPRINS.
 75. The method of claim 73 wherein said expanded nucleotiderepeat-containing RNA is any of SCA8/ataxin 8, ATN1/DRPLA, FMR1,AFF2/FMR2, frataxin/FXN, Htt, junctophilin-3 (JPH3), DMPK, zinc fingerprotein-9, Androgen receptor (AR) (X-linked), ataxin-1 (ATXN1), ATXN10,protein phosphatase PP2A (PPP2R2B), TATA box-binding protein (TBP),ATXN2, ATXN3, CACNA1A, ATXN7, and SCA8.
 76. The method of claim 72wherein said non-coding RNA is any of NEAT2, DMPK, U16, and U
 50. 77.The method of claim 70, wherein the oligonucleotide is a gapmer
 78. Themethod of claim 77, wherein the nucleobase sequence of theoligonucleotide is at least 95% complementary to the nuclear-retainedRNA as measured over the entirety of said oligonucleotide.
 79. Themethod of claim 77, wherein the nucleobase sequence of the modifiedoligonucleotide is 100% complementary to the nuclear-retained RNA asmeasured over the entirety of said modified oligonucleotide.
 80. Themethod of claim 77, wherein at least one internucleoside linkage of saidoligonucleotide is a modified internucleoside linkage.
 81. The method ofclaim 80, wherein each internucleoside linkage is a phosphorothioateinternucleoside linkage.
 82. The method of claim 70, wherein at leastone nucleoside of said oligonucleotide comprises a modified sugar. 83.The method of claim 82, wherein at least one modified sugar is abicyclic sugar.
 84. The method of claim 83, wherein at least onemodified sugar comprises a 2′-O-methoxyethyl, a 4′-CH(CH₃)—O-2′ bridge,or a 4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1 or
 2. 85. The method ofclaim 70, wherein at least one nucleoside of said oligonucleotidecomprises a modified nucleobase.
 86. The method of claim 85, wherein themodified nucleobase is a 5-methylcytosine.
 87. The method of claim 70,wherein the oligonucleotide comprises: a gap segment consisting oflinked deoxynucleosides; a 5′ wing segment consisting of linkednucleosides; a 3′ wing segment consisting of linked nucleosides; whereinthe gap segment is positioned between the 5′ wing segment and the 3′wing segment and wherein each nucleoside of each wing segment comprisesa modified sugar.
 88. The method of claim 87, wherein the modifiedoligonucleotide consists of 20 linked nucleosides.
 89. The method ofclaim 87, wherein each modified sugar comprises a 2′-O-methoxyethylmodification.
 90. The compound of claim 89, wherein each internucleosidelinkage is a phosphorothioate internucleoside linkage.